TgCRND8 Mice Research Articles

Higher-order interactions between hippocampal CA1 neurons are disrupted in amnestic mice.

Across systems, higher-order interactions between components govern emergent dynamics. Here we tested whether contextual threat memory retrieval in mice relies on higher-order interactions between dorsal CA1 hippocampal neurons requiring learning-induced dendritic spine plasticity. We compared population-level Ca2+ transients as wild-type mice (with intact learning-induced spine plasticity and memory) and amnestic mice (TgCRND8 mice with high levels of amyloid-β and deficits in learning-induced spine plasticity and memory) were tested for memory. Using machine-learning classifiers with different capacities to use input data with complex interactions, our findings indicate complex neuronal interactions in the memory representation of wild-type, but not amnestic, mice. Moreover, a peptide that partially restored learning-induced spine plasticity also restored the statistical complexity of the memory representation and memory behavior in Tg mice. These findings provide a previously missing bridge between levels of analysis in memory research, linking receptors, spines, higher-order neuronal dynamics and behavior.

Evaluation of the biodistribution and preliminary safety profile of a novel brain-targeted manganese dioxide-based nanotheranostic system for Alzheimer’s disease

A novel brain-targeted and reactive oxygen species-activatable manganese dioxide containing nanoparticle system functionalized with anti-amyloid-β antibody (named aAβ-BTRA-NC) developed by our group has shown great promise as a highly selective magnetic resonance imaging (MRI) contrast agent for early detection and multitargeted disease-modifying treatment of Alzheimer’s disease (AD). To further evaluate the suitability of the formulation for future clinical application, we investigated the safety, biodistribution, and pharmacokinetic profile of aAβ-BTRA-NC in a transgenic TgCRND8 mouse AD model, wild type (WT) littermate, and CD-1 mice. Dose-ascending studies demonstrated that aAβ-BTRA-NC was well-tolerated by the animals up to 300 μmol Mn/kg body weight [b.w.], 3 times the efficacious dose for early AD detection without apparent adverse effects; Histopathological, hematological, and biochemical analyses indicated that a single dose of aAβ-BTRA-NC did not cause any toxicity in major organs. Immunotoxicity data showed that aAβ-BTRA-NC was safer than commercially available gadolinium-based MRI contrast agents at an equivalent dose of 100 μmol/kg b.w. of metal ions. Intravenously administered aAβ-BTRA-NC was taken up by main organs with the order of liver, kidneys, intestines, spleen, followed by other organs, and cleared after one day to one week post injection. Pharmacokinetic analysis indicated that the plasma concentration profile of aAβ-BTRA-NC followed a 2-compartmental model with faster clearance in the AD mice than in the WT mice. The results suggest that aAβ-BTRA-NC exhibits a strong safety profile as a nanotheranostic agent which warrants more robust preclinical development for future clinical applications.

Tianma-Gouteng pair ameliorates the cognitive deficits on two transgenic mouse models of Alzheimer's disease

Ethnopharmacological relevanceAlzheimer's disease (AD) is a progressive neurodegenerative disease. Tianma-Gouteng Pair (TGP), commonly prescribed as a pair-herbs, can be found in many Chinese medicine formulae to treat brain diseases. However, the neuroprotective effects and molecular mechanisms of TGP remained unexplored. Aim of the studyThis study investigated the difference between the TgCRND8 and 5 × FAD transgenic mice, the anti-AD effects of TGP, and underlying molecular mechanisms of TGP against AD through the two mouse models. MethodsBriefly, three-month-old TgCRND8 and 5 × FAD mice were orally administered with TGP for 4 and 6 months, respectively. Behavioral tests were carried out to determine the neuropsychological functions. Moreover, immunofluorescence and western blotting assays were undertaken to reveal the molecular mechanisms of TGP. ResultsAlthough TgCRND8 and 5 × FAD mice had different beta-amyloid (Aβ) burdens, neuroinflammation status, and cognition impairments, TGP exerted neuroprotective effects against AD in the two models. In detail, behavioral tests revealed that TGP treatment markedly ameliorated the anxiety-like behavior, attenuated the recognition memory deficits, and increased the spatial learning ability as well as the reference memory of TgCRND8 and 5 × FAD mice. Moreover, TGP treatment could regulate the beta-amyloid precursor protein (APP) processing by inhibiting the Aβ production enzymes such as β- and γ-secretases and activating Aβ degrading enzyme to reduce Aβ accumulation. In addition, TGP reduced the Aβ42 level, the ratio of Aβ42/Αβ40, Aβ accumulation, and tau hyperphosphorylation in both the 5 × FAD and TgCRND8 mouse models. Furthermore, TGP ameliorated neuroinflammation by decreasing the densities of activated microglia and astrocytes, and inhibiting the production of inflammatory cytokines. TGP upregulated the SIRT1 and AMPK, and downregulated sterol response element binding protein 2 (SREBP2) in the brain of TgCRND8 mice and deactivation of the EPhA4 and c-Abl in the brain tissues of 5 × FAD mice. ConclusionOur experiments for the first time revealed the neuroprotective effects and molecular mechanism of TGP on 5 × FAD and TgCRND8 transgenic mouse models of different AD stages. TGP decreased the level of Aβ aggregates, improved the tauopathy, and reduced the neuroinflammation by regulation of the SIRT1/AMPK/SREBP2 axis and deactivation of EPhA4/c-Abl signaling pathway in the brains of TgCRND8 and 5 × FAD mice, respectively. All these findings unequivocally confirmed that the TGP would be promising in developing into an anti-AD therapeutic pharmaceutical.

Discovery of Novel Ophthalmic Imaging Biomarkers for Neurodegenerative Disease

AbstractBackgroundThe retina is increasingly investigated as a site of Alzheimer’s Disease (AD) manifestation, with amyloid β (Aβ) plaques being found in the retina of AD and mild cognitively impaired (MCI) patients, however functional changes in the retina are poorly understood. Retinal imaging techniques can be utilized for neurodegenerative biomarkers, since the retina is the only CNS organ available for noninvasive tracking.MethodRetinal imaging techniques were using to compare the retina of wild‐type (WT) or AD transgenic mice containing the APP: Swedish and Indiana mutation expressed under the prion promoter (TgCRND8) at 20 and 30 weeks of age (n = 10 mice per genotype). Optical coherence tomography (OCT) for retinal thickness assessment, Fundus Autofluorescence Imaging (FAF) for aggregate visualization, and electroretinogram (ERG) for retinal neuronal electrical activity were performed. Aβ ELISA was also performed on retina and cortex to evaluate amyloid burden correlation.ResultMean retinal insoluble Aβ 1‐42 was significantly elevated in TgCRND8 mice relative to WT at 23 weeks (35 vs 4.2 pg/mL, P<0.05) and 42 weeks (77.9 vs 7.5 pg/mL, P<0.05), and correlated with insoluble cortical brain Aβ 1‐42 (Pearson’s r = 0.847, P = 0.001). Total retinal thickness was significantly higher in TgCRND8 mice relative to WT (233 µm vs 225 µm, P<0.01). Specifically, the retinal nerve fiber layer (RNFL) was thicker in TgCRND8 mice (28.5 vs 22.9 µm, P<0.01). FAF imaging revealed significant autofluorescent aggregate detection in TgCRND8 mice both in the RNFL (94.3 vs 59.8 spots, P<0.01) and outer plexiform layer (OPL) (87.8 vs 49.3 spots, P<0.01). Lastly ERG revealed reduced b wave amplitude in TgCRND8 mice relative to WT (197.45 vs 457.9 µV, P<0.001) and photopic negative response (PhNR) wave amplitude (8.2 µm vs 25.7 457.9 µV, P<0.001). These values are indicative of dysfunctional bipolar and retinal ganglion cell electrical activity.ConclusionTgCRND8 mice have functional changes in retina thickness, FAF lipofuscin deposition, and ERG, which may prove invaluable as biomarkers for pre‐clinical AD diagnosis, monitoring and drug efficacy trials.

Nascent Aβ42 Fibrillization in Synaptic Endosomes Precedes Plaque Formation in a Mouse Model of Alzheimer's-like β-Amyloidosis.

Accumulation of amyloid-β peptide (Aβ) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aβ aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aβ accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aβ-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aβ homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aβ accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aβ monomers, oligomers, and fibrils. Levels of all soluble Aβ species declined thereafter, as Aβ42 turned progressively insoluble and accumulated in Aβ-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aβ was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aβ42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aβ42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aβ accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aβ in endosomes and, in TgCRND8 mice, Aβ overproduction causes rapid loss of Aβ42 solubility that curtails ECE-mediated degradation.SIGNIFICANCE STATEMENT Deposition of aggregated Aβ in extracellular plaques is a defining feature of AD. Aβ aggregates also accumulate in synapses and may contribute to the profound synaptic loss and cognitive dysfunction typical of the disease. However, it is not clear whether synaptotoxic Aβ is mainly derived from plaques or if it is produced and aggregated locally, within affected synaptic compartments. Filling this knowledge gap is important for the development of an effective treatment for AD, as extracellular and intrasynaptic pools of Aβ may not be equally modulated by immunotherapies or other therapeutic approaches. In this manuscript, we provide evidence that Aβ aggregates building up in synapses are formed locally, within synaptic endosomes, because of disruptions in nascent Aβ proteostasis.

Gut dysbiosis aggravates cognitive deficits, amyloid pathology and lipid metabolism dysregulation in a transgenic mouse model of Alzheimer's disease

Gut dysbiosis, a well-known risk factor to triggers the progression of Alzheimer's disease (AD), is strongly associated with metabolic disturbance. Trimethylamine N-oxide (TMAO), produced in the dietary choline metabolism, has been found to accelerate neurodegeneration in AD pathology. In this study, the cognitive function and gut microbiota of TgCRND8 (Tg) mice of different ages were evaluated by Morris water maze task (MWMT) and 16S rRNA sequencing, respectively. Young pseudo germ-free (PGF) Tg mice that received faecal microbiota transplants from aged Tg mice and wild-type (WT) mice were selected to determine the role of the gut microbiota in the process of neuropathology. Excessive choline treatment for Tg mice was used to investigate the role of abnormal choline metabolism on the cognitive functions. Our results showed that gut dysbiosis, neuroinflammation response, Aβ deposition, tau hyperphosphorylation, TMAO overproduction and cyclin-dependent kinase 5 (CDK5)/transcription 3 (STAT3) activation occurred in Tg mice age-dependently. Disordered microbiota of aged Tg mice accelerated AD pathology in young Tg mice, with the activation of CDK5/STAT3 signaling in the brains. On the contrary, faecal microbiota transplantation from WT mice alleviated the cognitive deficits, attenuated neuroinflammation, Aβ deposition, tau hyperphosphorylation, TMAO overproduction and suppressed CDK5/STAT3 pathway activation in Tg mice. Moreover, excessive choline treatment was also shown to aggravate the cognitive deficits, Aβ deposition, neuroinflammation and CDK5/STAT3 pathway activation. These findings provide a novel insight into the interaction between gut dysbiosis and AD progression, clarifying the important roles of gut microbiota-derived substances such as TMAO in AD neuropathology.

Perinatal S-Adenosylmethionine Supplementation Represses PSEN1 Expression by the Cellular Epigenetic Memory of CpG and Non-CpG Methylation in Adult TgCRD8 Mice.

DNA methylation, the main epigenetic modification regulating gene expression, plays a role in the pathophysiology of neurodegeneration. Previous evidence indicates that 5'-flanking hypomethylation of PSEN1, a gene involved in the amyloidogenic pathway in Alzheimer's disease (AD), boosts the AD-like phenotype in transgenic TgCRND8 mice. Supplementation with S-adenosylmethionine (SAM), the methyl donor in the DNA methylation reactions, reverts the pathological phenotype. Several studies indicate that epigenetic signatures, driving the shift between normal and diseased aging, can be acquired during the first stages of life, even in utero, and manifest phenotypically later on in life. Therefore, we decided to test whether SAM supplementation during the perinatal period (i.e., supplementing the mothers from mating to weaning) could exert a protective role towards AD-like symptom manifestation. We therefore compared the effect of post-weaning vs. perinatal SAM treatment in TgCRND8 mice by assessing PSEN1 methylation and expression and the development of amyloid plaques. We found that short-term perinatal supplementation was as effective as the longer post-weaning supplementation in repressing PSEN1 expression and amyloid deposition in adult mice. These results highlight the importance of epigenetic memory and methyl donor availability during early life to promote healthy aging and stress the functional role of non-CpG methylation.

Soluble amyloid-β dimers are resistant to amyloid-β prion conversion in vivo suggesting antiprion properties.

According to the instructions for authors, short communications should be published without abstract.

Correction: Temporal and spatially controlled APP transgene expression using Cre-dependent alleles

There were errors in Disease Models & Mechanisms (2022) 15, dmm049330 (doi:10.1242/dmm.049330).The authors recently discovered that one of the Cre-dependent transgenic mouse lines used in the study is not correctly described within the article. The mouse line labelled as R26-APP in the paper was originally described as containing both the Swedish and Iberian mutations, but carries only the Iberian mutation. This was discovered by The Jackson Laboratory when they sequenced through the complete insert to confirm the allele before entering the line into their repository for distribution. The authors also discovered an unintended point mutation located just outside the Aβ domain, M647T by APP695 numbering (M722T by APP770 numbering). They propose that the model should be identified as R26-APPIbe rather than as R26-APP.A diagram of the allele that appears in Fig. 2A has been corrected:In addition, a section describing the cloning methods in the Materials and Methods has been updated. The corrected and original versions are shown below:‘Cloning of R26-LSL-hAPPIbe transgene’ section (corrected). Human APPIbe 695 amino acid cDNA was generated in two steps. The N-terminal 1592 nucleotides of huAPP695 were cut from pcDNA3.1-sAPPα (a gift from Hui Zheng, BCM) with HindIII and SacI and subcloned into the same sites of transfer plasmid L4440 (Addgene, #1654). The remaining fragments were amplified from a human cDNA library using a two-step PCR method. Both PCR products contained the Ibe mutation as well as an overlap region needed to serve as an extended template for the second PCR. The first C-terminal fragment was amplified using hAPP C-terminus forward primer 5′-AACGAAAACCACCGTGGAGCT-3′ and Ibe reverse primer 5′-AAGGTGATGACGAACACTGTCG-3′. The second C-terminal fragment was amplified by Ibe forward primer 5′-CGACAGTGTTCGTCATCACCTT-3′ and hAPP C-terminus reverse primer 5′-GCGCGGATATCCTAGTTCTGCATCTGCTCAAAG-3′. The complete hAPP695Ibe C-terminus was then amplified from a mixture of the two shorter products using hAPP CT forward primer and hAPP CT reverse primer. The purified huAPPIbe C-terminal PCR fragment was digested with EcoRV and SacI and ligated into the EcoRV/SacI sites of L4440-APP(1-1592), resulting in L4440-APPIbe. The complete huAPPIbe sequence was transferred into an intermediary expression plasmid, which was digested with MluI and FseI and cloned into an Ai3-derived ROSA26 targeting vector (Madisen et al., 2010) to generate the final transgene construct pJJ-hAPPIbe. The modified Ai3 targeting vector retained the original Ai3 Cre-dependent CAG-loxP-stop-loxP sequence but removed the PGK-DTA negative selection cassette and shortened the 3′ targeting arm to reduce the overall vector size. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).‘Cloning of R26-LSL-hAPPSwe/Ibe transgene’ section (original). Human APPSwe/Ibe 695 amino acid cDNA was amplified from the pBR322-APPSwe/Ind plasmid used to generate the Tg CRND8 mouse model (Chishti et al., 2001) (a gift from David Westaway, University of Alberta, Canada) with primers that converted the original Indiana mutation V717F into the Iberian mutation I716F (point mutation numbering is for the standard human APP 770 amino acid isoform). The N-terminus of APPSwe/Ibe 695 was amplified using the forward primer 5′-GGCCGGCCGCCACCATGCTGCCCGGTTTGGCACT-3′ and reverse primer 5′-AAGGTGATGACGAACACTGTCG-3′; the C-terminus was amplified using the forward primer 5′-CGACAGTGTTCGTCATCACCTT-3′ and reverse primer 5′-GCGCGACGCGTCTAGTTCTGCATCTGCTCAAAGAA-3′. The resulting hAPPSwe/Ibe fragments were combined by amplification using the forward primer 5′-GGCCGGCCGCCACCATGCTGCCCGGTTTGGCACT-3′ and reverse primer 5′-GCGCGACGCGTCTAGTTCTGCATCTGCTCAAAGAA-3′. The complete hAPPSwe/Ibe 695 cassette was digested with MluI and FseI, and cloned into an Ai3-derived ROSA26 targeting vector (Madisen et al., 2010) that had been cut with the same enzymes to generate the final transgene construct pJJ-hAPPSwe/Ibe. The modified Ai3 targeting vector retained the original Cre-dependent CAGloxP-stop-loxP sequence but removed the PGK-DTA negative selection cassette and shortened the 3′ targeting arm to reduce the overall vector size. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).Throughout the paper, where the text refers to R26-APP, R26-APPSwe/Ibe or ROSA26-CAG-LSL-APPSwe/Ibe, corrections have been made to R26-APPIbe or ROSA26-CAG-LSL-APPIbe. The model will still be distributed by The Jackson Laboratory as stock #037319, but using the line name R26-APPIbe. The study results and conclusions remain unaffected.Both the online full-text and PDF versions have been updated. The authors apologise to readers for these errors and any inconvenience they may have caused.

Patchouli alcohol attenuates the cognitive deficits in a transgenic mouse model of Alzheimer’s disease via modulating neuropathology and gut microbiota through suppressing C/EBPβ/AEP pathway

BackgroundAlzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by progressive cognitive dysfunctions and behavioral impairments. Patchouli alcohol (PA), isolated from Pogostemonis Herba, exhibits multiple pharmacological properties, including neuroprotective effects. This study aimed to investigate the therapeutic effects of PA against AD using the TgCRND8 transgenic AD mouse model, and to explore the underlying mechanisms targeting CCAAT/enhancer-binding protein β/asparagine endopeptidase (C/EBPβ/AEP) signaling pathway.MethodsAfter genotyping to confirm the transgenicity, drug treatments were administered intragastrically once daily to 3-month-old TgCRND8 mice for 4 consecutive months. Several behavioral tests were applied to assess different aspects of neurological functions. Then the brain and colon tissues were harvested for in-depth mechanistic studies. To further verify whether PA exerts anti-AD effects via modulating C/EBPβ/AEP signaling pathway in TgCRND8 mice, adeno-associated virus (AAV) vectors encoding CEBP/β were bilaterally injected into the hippocampal CA1 region in TgCRND8 mice to overexpress C/EBPβ. Additionally, the fecal microbiota transplantation (FMT) experiment was performed to verify the potential role of gut microbiota on the anti-AD effects of PA.ResultsOur results showed that PA treatment significantly improved activities of daily living (ADL), ameliorated the anxiety-related behavioral deficits and cognitive impairments in TgCRND8 mice. PA modulated the amyloid precursor protein (APP) processing. PA also markedly reduced the levels of beta-amyloid (Aβ) 40 and Aβ42, suppressed Aβ plaque burdens, inhibited tau protein hyperphosphorylation at several sites and relieved neuroinflammation in the brains of TgCRND8 mice. Moreover, PA restored gut dysbiosis and inhibited the activation of the C/EBPβ/AEP signaling pathway in the brain and colon tissues of TgCRND8 mice. Interestingly, PA strikingly alleviated the AD-like pathologies induced by the overexpression of C/EBPβ in TgCRND8 mice. Additionally, the FMT of fecal microbiota from the PA-treated TgCRND8 mice significantly alleviated the cognitive impairments and AD-like pathologies in the germ-free TgCRND8 mice.ConclusionAll these findings amply demonstrated that PA could ameliorate the cognitive deficits in TgCRND8 mice via suppressing Aβ plaques deposition, hyperphosphorylation of tau protein, neuroinflammation and gut dysbiosis through inhibiting the activation of C/EBPβ/AEP pathway, suggesting that PA is a promising naturally occurring chemical worthy of further development into the pharmaceutical treatment of AD.

Sulforaphene, a CDK5 Inhibitor, attenuates cognitive deficits in a transgenic mouse model of Alzheimer's disease via reducing Aβ Deposition, tau hyperphosphorylation and synaptic dysfunction.

Alzheimer's disease (AD) is the most common form of neurodegenerative disorder characterized by progressive loss of memory and cognitive functions. There are two pathological hallmarks, including accumulation of amyloid plaques composed of β-amyloid peptide (Aβ) and deposits of neurofibrillatory tangles (NFT). Cyclin-dependent kinase 5 (CDK5), a serine/threonine kinase, plays an important role in synaptic plasticity and cognitive behavior. Sulforaphene (SF) has been demonstrated to exert anti-AD activity in AD rat model. In this study, we aimed to evaluate the cognitive deficits improving effects of SF on in TgCRND8 mice and to elucidate the underlying molecular mechanisms. TgCRND8 mice were intragastrically treated with SF (25 and 50mg/kg) for 4months from 3-month-old. The cognitive functions were assessed using Morris Water Maze Test. Cultured primary mouse neurons were pre-treated with SF, followed by co-treatment with Aβ1-42 oligomers. CDK5 inhibitor (roscovitine) was used to determine the involvement of CDK5/p25 pathway in the anti-AD effects of SF in primary neurons. Our results showed that SF treatment significantly ameliorated the cognitive deficits in TgCRND8 mice and protected primary mouse neurons against Aβ1-42 induced neurotoxicity. SF could modulate the expression of Aβ production related markers, and suppress the phosphorylation of tau protein at specific sites in the TgCRND8 mice. In addition, SF enhanced the expressions of synaptic plasticity related markers and CDK5. SF also markedly suppressed the CDK5/p25 activity. SF is a potent CDK5 inhibitor and a potential therapeutic agent for treatment and prevention of AD. Moreover, SF inhibited the overexpression of CDK5 in primary neurons of mouse.

Effect of Unloaded and Curcumin-Loaded Solid Lipid Nanoparticles on Tissue Transglutaminase Isoforms Expression Levels in an Experimental Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is a neurodegenerative disease representing the most prevalent cause of dementia. It is also related to the aberrant amyloid-beta (Aβ) protein deposition in the brain. Since oxidative stress is involved in AD, there is a possible role of antioxidants present in the effected person’s diet. Thus, we assessed the effect of the systemic administration of solid lipid nanoparticles (SLNs) to facilitate curcumin (CUR) delivery on TG2 isoform expression levels in Wild Type (WT) and in TgCRND8 (Tg) mice. An experimental model of AD, which expresses two mutated human amyloid precursor protein (APP) genes, was used. Behavioral studies were also performed to evaluate the improvement of cognitive performance and memory function induced by all treatments. The expression levels of Bcl-2, Cyclin-D1, and caspase-3 cleavage were evaluated as well. In this research, for the first time, we demonstrated that the systemic administration of SLNs-CUR, both in WT and in Tg mice, allows one to differently modulate TG2 isoforms, which act either on apoptotic pathway activation or on the ability of the protein to repair cellular damage in the brains of Tg mice. In this study, we also suggest that SLNs-CUR could be an innovative tool for the treatment of AD.

Connectomic analysis of Alzheimer's disease using percolation theory.

Alzheimer's disease (AD) is a severe neurodegenerative disorder that affects a growing worldwide elderly population. Identification of brain functional biomarkers is expected to help determine preclinical stages for targeted mechanistic studies and development of therapeutic interventions to deter disease progression. Connectomic analysis, a graph theory-based methodology used in the analysis of brain-derived connectivity matrices was used in conjunction with percolation theory targeted attack model to investigate the network effects of AD-related amyloid deposition. We used matrices derived from resting-state functional magnetic resonance imaging collected on mice with extracellular amyloidosis (TgCRND8 mice, n = 17) and control littermates (n = 17). Global, nodal, spatial, and percolation-based analysis was performed comparing AD and control mice. These data indicate a short-term compensatory response to neurodegeneration in the AD brain via a strongly connected core network with highly vulnerable or disconnected hubs. Targeted attacks demonstrated a greater vulnerability of AD brains to all types of attacks and identified progression models to mimic AD brain functional connectivity through betweenness centrality and collective influence metrics. Furthermore, both spatial analysis and percolation theory identified a key disconnect between the anterior brain of the AD mice to the rest of the brain network.

MORPHIOUS: an unsupervised machine learning workflow to detect the activation of microglia and astrocytes

BackgroundIn conditions of brain injury and degeneration, defining microglial and astrocytic activation using cellular markers alone remains a challenging task. We developed the MORPHIOUS software package, an unsupervised machine learning workflow which can learn the morphologies of non-activated astrocytes and microglia, and from this information, infer clusters of microglial and astrocytic activation in brain tissue.MethodsMORPHIOUS combines a one-class support vector machine with the density-based spatial clustering of applications with noise (DBSCAN) algorithm to identify clusters of microglial and astrocytic activation. Here, activation was triggered by permeabilizing the blood–brain barrier (BBB) in the mouse hippocampus using focused ultrasound (FUS). At 7 day post-treatment, MORPHIOUS was applied to evaluate microglial and astrocytic activation in histological tissue. MORPHIOUS was further evaluated on hippocampal sections of TgCRND8 mice, a model of amyloidosis that is prone to microglial and astrocytic activation.ResultsMORPHIOUS defined two classes of microglia, termed focal and proximal, that are spatially adjacent to the activating stimulus. Focal and proximal microglia demonstrated activity-associated features, including increased levels of ionized calcium-binding adapter molecule 1 expression, enlarged soma size, and deramification. MORPHIOUS further identified clusters of astrocytes characterized by activity-related changes in glial fibrillary acidic protein expression and branching. To validate these classifications following FUS, co-localization with activation markers were assessed. Focal and proximal microglia co-localized with the transforming growth factor beta 1, while proximal astrocytes co-localized with Nestin. In TgCRND8 mice, microglial and astrocytic activation clusters were found to correlate with amyloid-β plaque load. Thus, by only referencing control microglial and astrocytic morphologies, MORPHIOUS identified regions of interest corresponding to microglial and astrocytic activation.ConclusionsOverall, our algorithm is a reliable and sensitive method for characterizing microglial and astrocytic activation following FUS-induced BBB permeability and in animal models of neurodegeneration.

Ultrasound delivery of a TrkA agonist confers neuroprotection to Alzheimer-associated pathologies.

Early degeneration of basal forebrain cholinergic neurons contributes substantially to cognitive decline in Alzheimer’s disease. Evidence from preclinical models of neuronal injury and aging support a pivotal role for nerve growth factor (NGF) in neuroprotection, resilience, and cognitive function. However, whether NGF can provide therapeutic benefit in the presence of Alzheimer’s disease-related pathologies still unresolved. Perturbations in the NGF signalling system in Alzheimer’s disease may render neurons unable to benefit from NGF administration. Additionally, challenges related to brain delivery remain for clinical translation of NGF-based therapies in Alzheimer’s disease. To be safe and efficient, NGF-related agents should stimulate the NGF receptor, tropomyosin receptor kinase A (TrkA), avoid activation through the p75 neurotrophin receptor (p75NTR), and be delivered non-invasively to targeted brain areas using real-time monitoring. We addressed these limitations using MRI-guided focused ultrasound (MRIgFUS) to increase blood–brain barrier permeability locally and transiently, allowing an intravenously administered TrkA agonist that does not activate p75NTR, termed D3, to enter targeted brain areas. Here, we report the therapeutic potential of selective TrkA activation in a transgenic mouse model that recapitulates numerous Alzheimer’s disease-associated pathologies. Repeated MRIgFUS-mediated delivery of D3 (D3/FUS) improved cognitive function in the TgCRND8 model of Alzheimer’s disease. Mechanistically, D3/FUS treatment effectively attenuated cholinergic degeneration and promoted functional recovery. D3/FUS treatment also resulted in widespread reduction of brain amyloid pathology and dystrophic neurites surrounding amyloid plaques. Furthermore, D3/FUS markedly enhanced hippocampal neurogenesis in TgCRND8 mice, implicating TrkA agonism as a novel therapeutic target to promote neurogenesis in the context of Alzheimer’s disease-related pathology. Thus, this study provides evidence that selective TrkA agonism confers neuroprotection to effectively counteract Alzheimer’s disease-related vulnerability. Recent clinical trials demonstrate that non-invasive blood–brain barrier modulation using MRIgFUS is safe, feasible and reversible in Alzheimer’s disease patients. TrkA receptor agonists coupled with MRIgFUS delivery constitute a promising disease-modifying strategy to foster brain health and counteract cognitive decline in Alzheimer’s disease.

Combinatorial model of amyloid β and tau reveals synergy between amyloid deposits and tangle formation.

To illuminate the pathological synergy between Aβ and tau leading to emergence of neurofibrillary tangles (NFT) in Alzheimer's disease (AD), here, we have performed a comparative neuropathological study utilising three distinctive variants of human tau (WT tau, P301L mutant tau and S320F mutant tau). Previously, in non-transgenic mice, we showed that WT tau or P301L tau does not form NFT while S320F tau can spontaneously aggregate into NFT, allowing us to test the selective vulnerability of these different tau conformations to the presence of Aβ plaques. We injected recombinant AAV-tau constructs into neonatal APP transgenic TgCRND8 mice or into 3-month-old TgCRND8 mice; both cohorts were aged 3months post injection. This allowed us to test how different tau variants synergise with soluble forms of Aβ (pre-deposit cohort) or with frank Aβ deposits (post-deposit cohort). Expression of WT tau did not produce NFT or altered Aβ in either cohort. In the pre-deposit cohort, S320F tau induced Aβ plaque deposition, neuroinflammation and synaptic abnormalities, suggesting that early tau tangles affect the amyloid cascade. In the post-deposit cohort, contemporaneous expression of S320F tau did not exacerbate amyloid pathology, showing a dichotomy in Aβ-tau synergy based on the nature of Aβ. P301L tau produced NFT-type inclusions in the post-deposit cohort, but not in the pre-deposit cohort, indicating pathological synergy with pre-existing Aβ deposits. Our data show that different tau mutations representing specific folding variants of tau synergise with Aβ to different extents, depending on the presence of cerebral deposits.

Sex differences in the expression of synaptic proteins in the brain of the TgCRND8 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is progressive, neurodegenerative disorder, characterized by memory loss and cognitive decline. Advanced age and female sex are the most prominent risk factors. Although epidemiological and clinical studies have provided evidence for sex differences, relevant molecular mechanisms are not well characterized. To determine if overexpression of AD-related genes had any effect on synaptic proteins from either side of the synapse, the pre-synaptic marker, synaptophysin, and the postsynaptic marker, PSD-95, were analyzed in the hippocampus and cortex of old CRND8 mice and their respective controls. We hypothesized that synaptic proteins that have been shown to be reduced in post-mortem brain samples in AD and in animal AD models, would show a more pronounced pattern of changes in females than in males. Hippocampal and cortical samples of 14-mo TgCRND8 and control mice (male and female) were analyzed on the gradient gel for the presence of amyloid beta. Western blot experiments were used to determine protein levels of synaptophysin and PSD95 in these same brain regions. Mortality rates were higher in female TgCRND8 than in males compared to their controls. The presence of amyloid beta was confirmed in cortex and hippocampus of all TgCRND8 mice. Western blot results revealed significant decreases in synaptophysin and PSD95 in the hippocampus of TgCRND8 females compared to controls (*p<0.05) but not in males. In the cortex, synaptophysin and PSD95 were decreased only in male TgCRND8 mice, while PSD95 showed a trend in decrease in female TgCRND8 mice. Our results confirm reports of decreased synaptic proteins in AD mouse brain, but we further show that these changes are region- and sex-dependent.

Loss of an ER component, membralin, in astrocytes triggers neuroinflammation.

During the progression of Alzheimer's disease (AD), and other neurodegenerative disorders, glial cells including astrocytes and microglia undergo morphological and physiological changes indicative of reactivity. However, whether glial activation is a non-consequential event from pathological stress or alternatively, whether glial activation can contribute to disease pathogenesis is unclear. Endoplasmic reticulum (ER) is the primary cellular organelle for the synthesis of transmembrane and secreted proteins. Previously, we identified an ER protein, membralin, as a novel component of the ER-associated degradation (ERAD) complex. Loss of membralin impaired homeostatic turnover of a key g-secretase complex subunit, nicastrin, resulting in increased g-secretase complex formation and activity, and aggravated AD pathology in TgCRND8 mice. Further, conditional deletion of membralin in astrocytes decreased expression of the excitatory amino acid transporter 2 (EAAT2) and induced excitotoxicity. Using adeno-associated viral (AAV) vectors to elevate membralin levels in the SOD1G93A ALS mouse model can significantly extend the lifespan of these animals. We use RNAseq, electron microscope, primary astrocyte culture, animal models in the study. Analysis of astroglial ER morphology using electron microscope shows that lose of membralin leads to dilated ER, which indicated elevation of ER stress. Transcriptomic analysis of motor cortex from astrocyte-specific membralin knockout animals reveals induction of reactive astrocyte profiles and disease-associated microglia (DAM) signatures. Further, membralin upregulation in astrocytes can alleviate neurotoxic (A1) astrocyte induction. Together, these results indicate that membralin is a critical neuroinflammation regulator. Modulation of astrocyte membralin levels may be promising as a neuroinflammatory suppressor in AD.

Vascular endothelial growth factor associated dissimilar cerebrovascular phenotypes in two different mouse models of Alzheimer's Disease

Vascular perturbations and cerebral hypometabolism are emerging as important components of Alzheimer's disease (AD). While various in vivo imaging modalities have been designed to detect changes of cerebral perfusion and metabolism in AD patients and animal models, study results were often heterogenous with respect to imaging techniques and animal models. We therefore evaluated cerebral perfusion and glucose metabolism of two popular transgenic AD mouse strains, TgCRND8 and 5xFAD, at 7 and 12 months-of-age under identical conditions and analyzed possible molecular mechanisms underlying heterogeneous cerebrovascular phenotypes. Results revealed disparate findings in these two strains, displaying important aspects of AD progression. TgCRND8 mice showed significantly decreased cerebral blood flow and glucose metabolism with unchanged cerebral blood volume (CBV) at 12 months-of-age whereas 5xFAD mice showed unaltered glucose metabolism with significant increase in CBV at 12 months-of-age and a biphasic pattern of early hypoperfusion followed by a rebound to normal cerebral blood flow in late disease. Finally, immunoblotting assays suggested that VEGF dependent vascular tone change may restore normoperfusion and increase CBV in 5xFAD.

Modulating innate immune activation states impacts the efficacy of specific A\u03b2 immunotherapy

IntroductionPassive immunotherapies targeting Aβ continue to be evaluated as Alzheimer’s disease (AD) therapeutics, but there remains debate over the mechanisms by which these immunotherapies work. Besides the amount of preexisting Aβ deposition and the type of deposit (compact or diffuse), there is little data concerning what factors, independent of those intrinsic to the antibody, might influence efficacy. Here we (i) explored how constitutive priming of the underlying innate activation states by Il10 and Il6 might influence passive Aβ immunotherapy and (ii) evaluated transcriptomic data generated in the AMP-AD initiative to inform how these two cytokines and their receptors’ mRNA levels are altered in human AD and an APP mouse model.MethodsrAAV2/1 encoding EGFP, Il6 or Il10 were delivered by somatic brain transgenesis to neonatal (P0) TgCRND8 APP mice. Then, at 2 months of age, the mice were treated bi-weekly with a high-affinity anti-Aβ1–16 mAb5 monoclonal antibody or control mouse IgG until 6 months of age. rAAV mediated transgene expression, amyloid accumulation, Aβ levels and gliosis were assessed. Extensive transcriptomic data was used to evaluate the mRNA expression levels of IL10 and IL6 and their receptors in the postmortem human AD temporal cortex and in the brains of TgCRND8 mice, the later at multiple ages.ResultsPriming TgCRND8 mice with Il10 increases Aβ loads and blocks efficacy of subsequent mAb5 passive immunotherapy, whereas priming with Il6 priming reduces Aβ loads by itself and subsequent Aβ immunotherapy shows only a slightly additive effect. Transcriptomic data shows that (i) there are significant increases in the mRNA levels of Il6 and Il10 receptors in the TgCRND8 mouse model and temporal cortex of humans with AD and (ii) there is a great deal of variance in individual mouse brain and the human temporal cortex of these interleukins and their receptors.ConclusionsThe underlying immune activation state can markedly affect the efficacy of passive Aβ immunotherapy. These results have important implications for ongoing human AD immunotherapy trials, as they indicate that underlying immune activation states within the brain, which may be highly variable, may influence the ability for passive immunotherapy to alter Aβ deposition.