Young 3xTg-AD Research Articles

Altered firing output of VIP interneurons and early dysfunctions in CA1 hippocampal circuits in the 3xTg mouse model of Alzheimer's disease.

Alzheimer's disease (AD) leads to progressive memory decline, and alterations in hippocampal function are among the earliest pathological features observed in human and animal studies. GABAergic interneurons (INs) within the hippocampus coordinate network activity, among which type 3 interneuron-specific (I-S3) cells expressing vasoactive intestinal polypeptide and calretinin play a crucial role. These cells provide primarily disinhibition to principal excitatory cells (PCs) in the hippocampal CA1 region, regulating incoming inputs and memory formation. However, it remains unclear whether AD pathology induces changes in the activity of I-S3 cells, impacting the hippocampal network motifs. Here, using young adult 3xTg-AD mice, we found that while the density and morphology of I-S3 cells remain unaffected, there were significant changes in their firing output. Specifically, I-S3 cells displayed elongated action potentials and decreased firing rates, which was associated with a reduced inhibition of CA1 INs and their higher recruitment during spatial decision-making and object exploration tasks. Furthermore, the activation of CA1 PCs was also impacted, signifying early disruptions in CA1 network functionality. These findings suggest that altered firing patterns of I-S3 cells might initiate early-stage dysfunction in hippocampal CA1 circuits, potentially influencing the progression of AD pathology.

Chronic inflammation with microglia senescence at basal forebrain: impact on cholinergic deficit in Alzheimer's brain haemodynamics.

Cholinergic innervation in the brain is involved in modulating neurovascular function including cerebral blood flow haemodynamics in response to neuronal activity. Cholinergic deficit is associated with pathophysiology in Alzheimer's disease, albeit the aetiology remains to be clarified. In the current study, neocortex cerebral blood flow response to acetylcholine was evaluated by Laser-Doppler Flowmetry (LDF) in 3xTgAD Alzheimer's disease model) and wild-type mice of two age groups. The peak of cerebral blood flow to acetylcholine (i.v.) from baseline levels (% ΔrCBF) was higher in young 3xTgAD versus in wild-type mice (48.35; 95% CI:27.03-69.67 versus 22.70; CI:15.5-29.91, P < 0.05); this was reversed in old 3xTgAD mice (21.44; CI:2.52-40.35 versus 23.25; CI:23.25-39). Choline acetyltransferase protein was reduced in neocortex, while cerebrovascular reactivity to acetylcholine was preserved in young 3×TgAD mice. This suggests endogenous acetylcholine deficit and possible cholinergic denervation from selected cholinergic nuclei within the basal forebrain. The early deposition of tauopathy moieties (mutant hTau and pTau181) and its coincidence in cholinergic cell clusters (occasionaly), were observed at the basal forebrain of 3xTgAD mice including substantia innominate, nucleus Basalis of Meynert and nucleus of horizontal limb diagonal band of Broca. A prominent feature was microglia interacting tauopathy and demonstrated a variety of morphology changes particularly when located in proximity to tauopathy. The microglia ramified phenotype was reduced as evaluated by the ramification index and Fractal analysis. Increased microglia senescence, identified as SASP (senescence-associated secretory phenotype), was colocalization with p16Ink4ɑ, a marker of irreversible cell-cycle arrest in old 3xTgAD versus wild-type mice (P = 0.001). The p16Ink4ɑ was also observed in neuronal cells bearing tauopathy within the basal forebrain of 3xTgAD mice. TNF-ɑ, the pro-inflammatory cytokine elevated persistently in microglia (Pearson's correlation coefficient = 0.62) and the loss of cholinergic cells in vulnerable basal forebrain environment, was indicated by image analysis in 3xTgAD mice, which linked to the cholinergic deficits in neocortex rCBF haemodynamics. Our study revealed the early change of CBF haemodynamics to acetylcholine in 3xTgAD model. As a major effector of brain innate immune activation, microglia SASP with age-related disease progression is indicative of immune cell senescence, which contributes to chronic inflammation and cholinergic deficits at the basal forebrain. Targeting neuroinflammation and senescence may mitigate cholinergic pathophysiology in Alzheimer's disease.

Age Related Changes in Muscle Mass and Force Generation in the Triple Transgenic (3xTgAD) Mouse Model of Alzheimer's Disease.

Emerging evidence suggests that patients with Alzheimer’s disease (AD) may show accelerated sarcopenia phenotypes. To investigate whether pathological changes associated with neuronal death and cognitive dysfunction also occur in peripheral motor neurons and muscle as a function of age, we used the triple transgenic mouse model of AD (3xTgAD mice) that carries transgenes for mutant forms of APP, Tau, and presenilin proteins that are associated with AD pathology. We measured changes in motor neurons and skeletal muscle function and metabolism in young (2 to 4 month) female control and 3xTgAD mice and in older (18–20 month) control and 3xTgAD female mice. In older 3xTgAD mice, we observed a number of sarcopenia-related phenotypes, including significantly fragmented and denervated neuromuscular junctions (NMJs) associated with a 17% reduction in sciatic nerve induced vs. direct muscle stimulation induced contractile force production, and a 30% decrease in gastrocnemius muscle mass. On the contrary, none of these outcomes were found in young 3xTgAD mice. We also measured an accumulation of amyloid-β (Aβ) in both skeletal muscle and neuronal tissue in old 3xTgAD mice that may potentially contribute to muscle atrophy and NMJ disruption in the older 3xTgAD mice. Furthermore, the TGF-β mediated atrophy signaling pathway is activated in old 3xTgAD mice and is a potential contributing factor in the muscle atrophy that occurs in this group. Perhaps surprisingly, mitochondrial oxygen consumption and reactive oxygen species (ROS) production are not elevated in skeletal muscle from old 3xTgAD mice. Together, these results provide new insights into the effect of AD pathological mechanisms on peripheral changes in skeletal muscle.

Age dependence of retinal vascular plexus attenuation in the triple transgenic mouse model of Alzheimer's disease

The influence of Alzheimer's disease (AD) progression and severity on the structural and functional integrity of the cerebral vasculature is well recognized. The retina is an extension of the brain; thus, changes in retinal vascular features may serve as markers of AD cerebrovascular pathologies. However, differentiating normal aging-versus AD-induced retinal vascular changes is unresolved. Therefore, we compared and quantified changes in superficial (SVP), intermediate (IVP), and deep (DVP) retinal vascular plexuses in young, middle-age, and old triple transgenic mouse model of AD (3xT-AD) to the changes that occur in age-matched controls (C57BL/6j). We used immunostaining combined with a novel tissue optical clearing approach along with a computational tool for quantitative analysis of vascular network alterations (vessel length and density) in SVP, IVP, and DVP. All three layers had comparable structural features and densities in young 3xTg-AD and control animals. In controls, IVP and DVP densities decreased with aging (−14% to −32% change from young to old, p < 0.05), while no changes were observed in SVP. In contrast, vascular parameters in the transgenic group decreased in all three layers with aging (−12% to −49% change from young to old, p < 0.05). Furthermore, in the old group, SVP and DVP vascular parameters were lower in the transgenics compared to age-matched controls (p < 0.05). Our analysis demonstrates that normal aging and progression of AD lead to various degrees of vascular alterations in the retina. Specifically, compared to normal aging, changes in vascular features of SVP and DVP regions of the retina are accelerated during AD progression. Considering recent advances in the field of depth-resolved imaging of retinal capillary network and microangiography, noninvasive quantitative monitoring of changes in retinal vascular network parameters of SVP and DVP may serve as markers for diagnosis and staging of Alzheimer's disease and discriminating AD-induced vascular attenuation from age-related vasculopathy.

Influence of fecal microbiota transplantation on gut microbiota composition and neuroinflammation of 3xTg-AD mice.

The gut microbiota, the aggregate of all microbial cells that inhabit the gut, bidirectionally communicates with the brain via cytokines, neurotransmitters, hormones, and secondary metabolites via the gut-brain axis. The gut microbiota is thought to contribute to the development of Alzheimer's disease (AD), characterized by plaque deposition, neurofibrillary tangles, and neuroinflammation. We hypothesize that manipulation of the gut microbiota can alter development of AD pathologies and neuroinflammation via the gut microbiota-brain axis. To further elucidate the role of the gut-brain axis in AD, we performed fecal microbiota transplants (FMT) from aged (52-64 weeks) 3xTg-AD mice, modeling plaques and neurofibrillary tangles, to young 3xTg-AD (n=5) or wild-type mice (n=10). Phosphate buffered saline (PBS) was gavaged into 3xTg-AD (n=5) and wild-type mice (n=10) as a control. For FMT, a fecal slurry from aged 3xTg-AD mice was prepared and given to experimental mice via oral gavage. At 8 weeks, mice were gavaged with FMT or PBS for 5 consecutive days, followed by fortnightly maintenance transplants for 24 weeks. The V4 region of the 16S rRNA gene was sequenced on the Illumina MiSeq. Data were analyzed using QIIME 2. Reverse transcriptase qPCR was used to assess microgliosis, astrocytosis, and Th1/Th2 inflammation in the hippocampus of the FMT cohort at 24 weeks of age. Our results show a shift in microbiome composition in FMT-treated mice when compared to control (PBS-treated) mice. Bacteroides were increased in 3xTg-AD and wild-type mice receiving FMT. At 24 weeks of age, there was no difference in neuroinflammation between mice treated with FMT compared to control (PBS) in 3xTg-AD or wild type mice. We observed partial engraftment of the gut microbiota from aged 3xTg-AD mice in all FMT-treated mice. We demonstrate the ability to transplant an aged gut microbiome into young mice, however we did not observe changes in neuroinflammation at 24 weeks of age. Future studies will evaluate neuroinflammation and neuropathologies at later time points. These studies will contribute to our understanding of how features of the gut microbiota may contribute to AD development.

Age-related impairment of cerebral blood flow response to KATP channel opener in Alzheimer’s disease mice with presenilin-1 mutation

Local cerebral blood flow (CBF) responses to neuronal activity are essential for cognition and impaired CBF responses occur in Alzheimer’s disease (AD). In this study, regional CBF (rCBF) responses to the KATP channel opener diazoxide were investigated in 3xTgAD, WT and mutant Presenilin 1(PS1M146V) mice from three age groups using Laser-Doppler flowmetry. The rCBF response was reduced early in young 3xTgAD mice and almost absent in old 3xTgAD mice, up to 30%–40% reduction with altered CBF velocity and mean arterial pressure versus WT mice. The impaired rCBF response in 3xTgAD mice was associated with progression of AD pathology, characterized by deposition of intracellular and vascular amyloid-β (Aβ) oligomers, senile plaques and tau pathology. The nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine abolished rCBF response to diazoxide suggesting NO was involved in the mediation of vasorelaxation. Levels of phosphor-eNOS (Ser1177) diminished in 3xTgAD brains with age, while the rCBF response to the NO donor sodium nitroprusside remained. In PS1M146V mice, the rCBF response to dizoxide reduced and high molecular weight Abeta oligomers were increased indicating PS1M146V contributed to the dysregulation of rCBF response in AD mice. Our study revealed an Aβ oligomer-associated compromise of cerebrovascular function in rCBF response to diazoxide in AD mice with PS1M146V mutation.

Accumulation of\xa0amyloid precursor protein C-terminal fragments triggers mitochondrial structure, function, and mitophagy defects in Alzheimer\u2019s disease models and human brains

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) could correspond to an etiological trigger of Alzheimer’s disease (AD) pathology. Altered mitochondrial homeostasis is considered an early event in AD development. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established. Here, we demonstrate in neuroblastoma SH-SY5Y cells expressing either APP Swedish mutations, or the β-secretase-derived APP-CTF fragment (C99) combined with β- and γ-secretase inhibition, that APP-CTFs accumulation independently of Aβ triggers excessive mitochondrial morphology alteration (i.e., size alteration and cristae disorganization) associated with enhanced mitochondrial reactive oxygen species production. APP-CTFs accumulation also elicit basal mitophagy failure illustrated by enhanced conversion of LC3, accumulation of LC3-I and/or LC3-II, non-degradation of SQSTM1/p62, inconsistent Parkin and PINK1 recruitment to mitochondria, enhanced levels of membrane and matrix mitochondrial proteins, and deficient fusion of mitochondria with lysosomes. We confirm the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo in young 3xTgAD transgenic mice treated with γ-secretase inhibitor as well as in adeno-associated-virus-C99 injected mice. Comparison of aged 2xTgAD and 3xTgAD mice indicates that, besides APP-CTFs, an additional contribution of Aβ to late-stage mitophagy activation occurs. Importantly, we report on mitochondrial accumulation of APP-CTFs in human post-mortem sporadic AD brains correlating with mitophagy failure molecular signature. Since defective mitochondria homeostasis plays a pivotal role in AD pathogenesis, targeting mitochondrial dysfunctions and/or mitophagy by counteracting early APP-CTFs accumulation may represent relevant therapeutic interventions in AD.

Treatment with scFv-h3D6 Prevented Neuronal Loss and Improved Spatial Memory in Young 3xTg-AD Mice by Reducing the Intracellular Amyloid-β Burden.

The intracellular deposition of amyloid-β (Aβ) peptides has been described in the brains of both Alzheimer's disease (AD) patients and animal models. A correlation between the intracellular amyloid burden and neurodegeneration has recently been reported in a triple-transgenic AD (3xTg-AD) murine model. In the present study, we assessed the effect of scFv-h3D6, an anti-Aβ single-chain variable fragment (scFv) derived from the antibody bapineuzumab, on amyloid pathology in 5-month-old 3xTg-AD female mice, focusing on intracellular Aβ clearance, neuronal survival, and functional abilities. We also examined neuroinflammation and the histology of peripheral organ samples to detect any adverse effects. A single intraperitoneal injection of scFv-h3D6 dramatically reduced intracellular Aβ burden in the deep layers of the cerebral cortex, pyramidal cells layer of the hippocampus, and basolateral amygdalar nucleus. The treatment prevented neuronal loss in the hippocampus and amygdala, while neither astrogliosis nor microgliosis was induced. Instead, an increase in the size of the white pulp after the treatment indicated that the spleen could be involved in the clearance mechanism. Although the treatment did not ameliorate behavioral and psychological symptoms of dementia-like symptoms, the results of cognitive testing pointed to a noticeable improvement in spatial memory. These findings indicated that the mechanism underlying the therapeutic effect of scFv-h3D6 was the clearance of intracellular Aβ, with subsequent prevention of neuronal loss and amelioration of cognitive disabilities. The treatment was safe in terms of neuroinflammation and kidney and liver function, whereas some effects on the spleen were observed.

LOSS OF BILIVERDIN REDUCTASE-A FAVORS TAU HYPERPHOSPHORYLATION IN ALZHEIMER DISEASE

Hyper-active GSK-3β favors Tau phosphorylation during the progression of Alzheimer disease (AD). Akt is one of the main kinases inhibiting GSK-3β and its activation occurs in response to neurotoxic stimuli including, i.e., oxidative stress. Biliverdin reductase-A (BVR-A) is a scaffold protein favoring the Akt-mediated inhibition of GSK-3β. Interestingly, reduced BVR-A levels along with increased oxidative stress were observed early in the hippocampus of 3xTg-AD mice (at 6 months), thus suggesting that loss of BVR-A could be a limiting factor in the oxidative stress-induced Akt-mediated inhibition of GSK-3β in AD and would favor increased Tau hyperphosphorylation. We evaluated changes of BVR-A, Akt, GSK-3β, oxidative stress and Tau phosphorylation levels (1) in young (6-months) and old (12-months) 3xTg-AD mice; and (2) in post-mortem inferior parietal lobule (IPL) samples from MCI, AD and age-matched controls. Furthermore, similar analyses were performed in vitro in HEK cells lacking BVR-A and treated with H2O2. Reduced BVR-A levels along with (1) increased oxidative stress; (2) reduced GSK-3β inhibition and (3) increased Tau Ser404 (target of GSK-3β activity) phosphorylation without changes of Akt activation both in young mice and in MCI, were observed. Interestingly, cells lacking BVR-A and treated with H2O2 showed reduced GSK-3β inhibition and increased Tau Ser404 phosphorylation, which resulted from a defect of Akt and GSK-3β physical interaction. Furthermore, reduced levels of Akt/GSK-3β complex were confirmed in the hippocampus of young 3xTg-AD mice and human MCI brain. In our study we demonstrate that loss of BVR-A impairs the neuroprotective Akt-mediated inhibition of GSK-3β in response to oxidative stress, thus contributing to Tau hyper-phosphorylation in early stage AD. This molecular mechanism shed a light on involvement of BVR-A as a scaffold in Tauopathy at early AD progression.

Loss of biliverdin reductase-A favors Tau hyper-phosphorylation in Alzheimer's disease

Hyper-active GSK-3β favors Tau phosphorylation during the progression of Alzheimer's disease (AD). Akt is one of the main kinases inhibiting GSK-3β and its activation occurs in response to neurotoxic stimuli including, i.e., oxidative stress. Biliverdin reductase-A (BVR-A) is a scaffold protein favoring the Akt-mediated inhibition of GSK-3β. Reduced BVR-A levels along with increased oxidative stress were observed early in the hippocampus of 3xTg-AD mice (at 6 months), thus suggesting that loss of BVR-A could be a limiting factor in the oxidative stress-induced Akt-mediated inhibition of GSK-3β in AD.We evaluated changes of BVR-A, Akt, GSK-3β, oxidative stress and Tau phosphorylation levels: (a) in brain from young (6-months) and old (12-months) 3xTg-AD mice; and (b) in post-mortem inferior parietal lobule (IPL) samples from amnestic mild cognitive impairment (MCI), from AD and from age-matched controls. Furthermore, similar analyses were performed in vitro in cells lacking BVR-A and treated with H2O2.Reduced BVR-A levels along with: (a) increased oxidative stress; (b) reduced GSK-3β inhibition; and (c) increased Tau Ser404 phosphorylation (target of GSK-3β activity) without changes of Akt activation in young mice, were observed. Similar findings were obtained in MCI, consistent with the notion that this is a molecular mechanism disrupted in humans. Interestingly, cells lacking BVR-A and treated with H2O2 showed reduced GSK-3β inhibition and increased Tau Ser404 phosphorylation, which resulted from a defect of Akt and GSK-3β physical interaction. Reduced levels of Akt/GSK-3β complex were confirmed in both young 3xTg-AD and MCI brain.We demonstrated that loss of BVR-A impairs the neuroprotective Akt-mediated inhibition of GSK-3β in response to oxidative stress, thus contributing to Tau hyper-phosphorylation in early stage AD. Such changes potential provide promising therapeutic targets for this devastating disorder.

Probing the putative α7 nAChR/NMDAR complex in human and murine cortex and hippocampus: Different degrees of complex formation in healthy and Alzheimer brain tissue.

α7 nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartate receptors (NMDARs) are key mediators of central cholinergic and glutamatergic neurotransmission, respectively. In addition to numerous well-established functional interactions between α7 nAChRs and NMDARs, the two receptors have been proposed to form a multimeric complex, and in the present study we have investigated this putative α7 nAChR/NMDAR assembly in human and murine brain tissues. By α-bungarotoxin (BGT) affinity purification, α7 and NMDAR subunits were co-purified from human and murine cortical and hippocampal homogenates, substantiating the notion that the receptors are parts of a multimeric complex in the human and rodent brain. Interestingly, the ratios between GluN1 and α7 levels in BGT pull-downs from cortical homogenates from Alzheimer’s disease (AD) brains were significantly lower than those in pull-downs from non-AD controls, indicating a reduced degree of α7 nAChR/NMDAR complex formation in the diseased tissue. A similar difference in GluN1/α7 ratios was observed between pull-downs from cortical homogenates from adult 3xTg-AD and age-matched wild type (WT) mice, whereas the GluN1/α7 ratios determined in pull-downs from young 3xTg-AD and age-matched WT mice did not differ significantly. The observation that pretreatment with oligomeric amyloid-β1–42 reduced GluN1/α7 ratios in BGT pull-downs from human cortical homogenate in a concentration-dependent manner provided a plausible molecular mechanism for this observed reduction. In conclusion, while it will be important to further challenge the existence of the putative α7 nAChR/NMDAR complex in future studies applying other methodologies than biochemical assays and to investigate the functional implications of this complex for cholinergic and glutamatergic neurotransmission, this work supports the formation of the complex and presents new insights into its regulation in healthy and diseased brain tissue.

EFFECT OF ISOFLURANE ON NEUROPATHOLOGY AND COGNITIVE IMPAIRMENT IN THE TRIPLE TRANSGENIC ALZHEIMER’S MOUSE MODEL

This study examines the association between isoflurane mediated effects on neuropathology, cognitive performance, and synaptic physiology in early postnatal and aged triple transgenic Alzheimer's disease (3xTg-AD) mice. Male and female C57BL6/J (NonTg) and 3xTg-AD mice (age: PND 7 and 14+ months) were anesthetized with isoflurane in oxygen/air for 2 hours each day over five consecutive days. Mice from each strain and age groups exposed to just oxygen/air served as controls. Plasma levels of S100β 6 hrs after anesthesia were elevated in both NonTg and 3xTg-AD, which was more profound in the 3xTg-AD mice. Isoflurane increased the number of positive neurons for Aβ deposition and oligomers in 3xTg-AD mice. Morris Water Maze (MWM) test showed that young NonTg and 3xTg-AD mice were slow to learn during reference learning test and performed poorly in short-term and working memory tests compared to age-matched controls. Young 3xTg-AD treated with isoflurane performed significantly worse in these spatial learning tests than the isoflurane-treated NonTg mice. Cognitive impairments in the aged mice were noted in short-term memory test in the aged 3xTg-AD mice and in both strains during the long-term and working memory tests. Extracellular field recordings showed hyperexcitable basal synaptic transmission, unstable LTP, and reduced paired pulse facilitation (PPF) in untreated aged 3xTg-AD mice compared to NonTg mice. Isoflurane depressed basal synaptic transmission, more pronounced in the aged 3xTg-AD group. PPF was significantly reduced in both young and old NonTg and 3xTg-AD mice following isoflurane, but more profound in aged 3xTg-AD mice. Isoflurane impaired LTP in young 3xTg and in both young and aged NonTg mice as reflected in the lack of change in extracellular field potential (fEPSP) slope from baseline. Isoflurane normalized and potentiated fEPSP slope to control levels in aged 3xTg mice . Acute treatment of isoflurane to ex-vivo hippocampal slices from young and aged NonTg and 3xTg-AD mice previously exposed to the same anesthetic in vivo suppressed LTP. Acute application of isoflurane to slices from aged 3xTg-AD mice previously exposed to iso displayed severely reduced LTP. isoflurane-mediated neuropathologic and cognitive defects in AD mice are associated with synaptic pathologies in an age-dependent manner.

BILIVERDIN REDUCTASE-A MEDIATES THE BENEFICIAL EFFECTS OF INTRANASAL INSULIN ADMINISTRATION ON AD PATHOLOGY IN THE BRAIN OF 3XTG-AD MICE

Biliverdin reductase-A (BVR-A) is a novel direct target of the insulin receptor, which phosphorylate BVR-A activating its Ser/Thr/Tyr kinase activity. Through this activity, BVR-A negatively regulates IRS1 activation, thus allowing the correct transduction of the insulin-mediated signaling. Along the progression of AD pathology we identified two phases in which: (1) the early impairment of BVR-A is responsible for the hyper-activation of IRS1, which then (2) causes the stimulation of feedback mechanisms including mTOR, aimed to turn-off IRS1 hyper-activity (Fig.1), thus promoting brain insulin resistance (b.i.r.). Reduced BVR-A activity is therefore an early event triggering the onset b.i.r̤ Intranasal insulin (I-Ins) administration is under evaluation as therapeutic strategy to alleviate b.i.r. in AD. However, the exact molecular mechanisms underlying I-Ins beneficial effects are still unclear. The goal of our project was to clarify whether the I-Ins-associated beneficial effects were mediated by the restoration of BVR-A activity. Changes of (1) the insulin signaling machinery (IR/IRS1/ ERK1/2/AKT/mTOR levels and activation) (2) total OS markers (PC, HNE, 3-NT) and (3) Aβ and tau levels, were evaluated in the hippocampus and cortex of 3xTg-AD and WT mice undergoing an early (4 months) or late (10 moths) I-Ins treatment (1 U/day, 3 times per week, for 2 months) (Fig.2). The morris water maze (MWM) and the novel object recognition (NOR) tasks were used to test cognitive functions. Cell-based experiments to support in vivo data were performed in HEK-APPSwe cells. I-Ins administration rescues the activation of BVR-A both in young and old 3xTg-AD mice. Improved BVR-A activity is associated with (1) a restoration of the insulin signaling cascade, (2) reduced OS markers and (3) a reduction of Tau pathology. All these changes parallel an improved cognition (Fig.3). Cell-based experiments confirmed the central role of BVR-A by showing that the effects of insulin are abolished when BVR-A is knocked-down. Our data highlight that BVR-A plays a pivotal role in the regulation of the insulin signaling in the brain. Restoration of BVR-A activity first, sheds light on the molecular mechanisms underlie I-Ins-mediated beneficial effects, and then suggest the role of BVR-A as potential therapeutic target to prevent b.i.r.in AD. (A) Proposed mechanism leading to b.i.r. in AD. Under physiological condition BVR-A regulates the acitvation of IRS1, thus controlling the correct transduction of insulin signaling cascade. In 3xTg-AD mice, an early impairment of BVR-A (6 months) is responsible for the hyper-activation of IRS1, which sustains the activation of the insulin signaling cascade for a time longer than normal (6 months). This phenomenon is aggravated by the further rise of the oxidative/nitrosative stress levels (PC, HNE and 3-NT), which results in the oxidative stress-induced impairment of BVR-A, which finally contributes to maintain IRS1 hyperactive. Persistence of IRS1 hyper-activation represents a signal for the activation of feedback mechanisms including mTOR, which at the end is responsible for the inhibitory phospshorylation of IRS1 and thus for the onset of brain insulin resistance (b.i.r.) (12 months). (B) Temporal profile of the events promoting BIR in the hippocampus 3×Tg-AD mice. Arrows, promotion; dotted lines, inhibition; Y, phosphor-Tyr residues; S, phospho-Ser residues; Y-NO2, 3-NT modifications. Scheme of the treatment used in this project. Intranasal insulin (I-Ins) administration prevents the impairment of BVR-A and the hyper-activation of IRS1. Normalization of the activity of the BVR-A/IRS1 axis is associated the correct transduction of the insulin signaling cascade, which finally prevents the cognitive decline observed in the 3xTg-AD mice.

Water and T-maze protocols are equally efficient methods to assess spatial memory in 3xTg Alzheimer’s disease mice

Rodent spatial memory is commonly tested using the water-maze; however, there is a potential confound of stress on learning in this behavioural paradigm. This is particularly relevant when testing spatial memory in models of neurodegeneration, such as the 3xTg mouse model for Alzheimer’s disease. Here, we first confirmed that 3xTgAD mice express fear conditioning and then compared the performance of young and middle-aged mice on short-duration versions of the radial arm water-maze (RAWM) and the minimally stressful T-maze spontaneous alternation task. Our main questions were: (1) does the reliance on stressors in water-maze training mask the true cognitive ability of 3xTgAD mice; and (2) are 3xTgAD mice similarly impaired in water-maze and T-maze protocols. Firstly, male and female 3xTgAD mice displayed intact freezing responses in both contextual and Pavlovian fear conditions. As male 3xTgAD mice displayed relatively enhanced fear responses the remaining tests were performed using only female 3xTgAD and control mice in order to equate for response to stressors. We found that alternation rates after both short and long delays were impaired at both ages in female 3xTgAD mice, indicative of robust spatial working memory deficits. For RAWM, again performance deficits were found in young 3xTgAD mice. As both tasks had similar efficacy at revealing early spatial memory deficits, we suggest that spontaneous behavioural protocols be prioritised over water maze testing in models such the 3xTgAD mouse as the former provide a far less stressful but equally effective alternative.

371 - Improvement of BVR-A Activity Ameliorates Brain Insulin Resistance in Alzheimer Disease Following Intranasal Insulin Administration

Brain insulin resistance (b.i.r.) was proposed as a mechanistic mediator of the cognitive deficits observed in Alzheimer disease (AD). Interestingly, the efficacy of intranasal insulin (I-Ins) administration – which allows to directly transport insulin to the CNS – was proposed as an innovative therapeutic strategy to alleviate cognitive dysfunction in AD. However, significant gaps in our knowledge about the mechanisms underlying these beneficial outcomes still exist. We previously reported about the oxidative-stress (OS) induced impairment of biliverdin reductase A (BVR-A) [a novel direct target of the insulin receptor (IR) kinase activity] as an early event contributing to b.i.r. in AD. Indeed, under normal conditions, once IR-activated BVR-A controls the activation of the insulin receptor substrate (IRS)-1, thus representing an upstream regulator of the insulin signaling cascade. Conversely, along the progression of AD pathology, the OS-induced inactivation of BVR-A promotes the hyper-activation of the IR/IRS-1 axis, which finally lead to b.i.r.. Here we hypothesized that the I-Ins-associated beneficial effects on cognition would be mediated by the restoration of BVR-A activity. To this aim, we evaluated in vivo (i) changes of the insulin signaling cascade (ii) variations of total OS markers (PC, HNE, 3-NT), (iii) changes of AD neuropathology markers e.g., Aβ and tau levels, in the hippocampus of 3xTg-AD and WT mice undergoing an early (4 months) or late (10 moths) intranasal insulin treatment (1 U/day, 3 times per week, for 2 months). Furthermore, changes of cognitive functions assessed through the morris water maze (MWM) and the novel object recognition (NOR) tasks, were evaluated. We found that I-Ins administration rescue the activation of BVR-A both in young and old 3xTg-AD mice. Improved BVR-A activity is associated with (i) reduced OS markers, (ii) a restoration of the insulin signaling cascade and (iii) a reduction of Tau pathology in the hippocampus. All these changes parallel an improved cognition. Interestingly, cell-based experiments confirmed the role of BVR-A by showing no beneficial effects of insulin when BVR-A is knocked-down.

Chronic cerebral hypoperfusion enhances Tau hyperphosphorylation and reduces autophagy in Alzheimer's disease mice.

Cerebral hypoperfusion and impaired autophagy are two etiological factors that have been identified as being associated with the development of Alzheimer’s disease (AD). Nevertheless, the exact relationships among these pathological processes remain unknown. To elucidate the impact of cerebral hypoperfusion in AD, we created a unilateral common carotid artery occlusion (UCCAO) model by occluding the left common carotid artery in both young and old 3xTg-AD mice. Two months after occlusion, we found that ligation increases phospho-Tau (p-Tau) at Serine 199/202 in the hippocampus of 3-month-old AD mice, compared to sham-operated AD mice; whereas, there is no change in the wild type (WT) mice after ligation. Moreover, cerebral hypoperfusion led to significant increase of p-Tau in both the hippocampus and cortex of 16-month-old AD mice and WT mice. Notably, we did not detect any change in Aβ42 level in either young or old AD and WT mice after ligation. Interestingly, we observed a downregulation of LC3-II in the cortex of aged AD mice and WT mice after ligation. Our results suggest that elevated p-Tau and reduced autophagy are major cellular changes that are associated with hypoperfusion in AD. Therefore, targeting p-Tau and autophagy pathways may ameliorate hypoperfusion-induced brain damage in AD.

Allopregnanolone preclinical acute pharmacokinetic and pharmacodynamic studies to predict tolerability and efficacy for Alzheimer's disease.

To develop allopregnanolone as a therapeutic for Alzheimer’s disease, we investigated multiple formulations and routes of administration in translationally relevant animal models of both sexes. Subcutaneous, topical (transdermal and intranasal), intramuscular, and intravenous allopregnanolone were bolus-administered. Pharmacokinetic analyses of intravenous allopregnanolone in rabbit and mouse indicated that peak plasma and brain levels (3-fold brain/plasma ratios) at 5min were sufficient to activate neuroregenerative responses at sub-sedative doses. Slow-release subcutaneous suspension of allopregnanolone displayed 5-fold brain/plasma ratio at Cmax at 30min. At therapeutic doses by either subcutaneous or intravenous routes, allopregnanolone mouse plasma levels ranged between 34-51ng/ml by 30min, comparable to published endogenous human level in the third trimester of pregnancy. Exposure to subcutaneous, topical, intramuscular, and intravenous allopregnanolone, at safe and tolerable doses, increased hippocampal markers of neurogenesis including BrdU and PCNA in young 3xTgAD and aged wildtype mice. Intravenous allopregnanolone transiently and robustly phosphorylated CREB within 5min and increased levels of neuronal differentiation transcription factor NeuroD within 4h. Neurogenic efficacy was achieved with allopregnanolone brain exposure of 300-500hr*ng/g. Formulations were tested to determine the no observable adverse effect level (NOAEL) and maximally tolerated doses (MTD) in male and female rats by sedation behavior time course. Sex differences were apparent, males exhibited ≥40% more sedation time compared to females. Allopregnanolone formulated in sulfobutyl-ether-beta-cyclodextrin at optimized complexation ratio maximized allopregnanolone delivery and neurogenic efficacy. To establish the NOAEL and MTD for Allo-induced sedation using a once-per-week intravenous regenerative treatment regimen: In female rats the NOAEL was 0.5mg/kg and MTD 2mg/kg. The predicted MTD in human female is 0.37mg/kg. In male rats the NOAEL and MTD were less than those determined for female. Outcomes of these PK/PD studies predict a safe and efficacious dose range for initial clinical trials of allopregnanolone for Alzheimer’s disease. These findings have translational relevance to multiple neurodegenerative conditions.

Impaired Src signaling and post-synaptic actin polymerization in Alzheimer's disease mice hippocampus — Linking NMDA receptors and the reelin pathway

Early cognitive deficits in Alzheimer's disease (AD) have been related to deregulation of N-methyl-d-aspartate receptors (NMDARs) and synaptic dysfunction in response to amyloid-beta peptide. NMDAR anchorage to post-synaptic membrane depends in part on Src kinase, which is also implicated in NMDAR activation and actin cytoskeleton stabilization, two processes relevant for normal synaptic function. In this study we analyzed the changes in GluN2B subunit phosphorylation and the levels of proteins involved in Src related signaling pathways linking the Tyr kinase to actin cytoskeleton polymerization, namely reelin, disabled-1 (Dab1) and cortactin, in hippocampal and cortical homogenates obtained from the triple transgenic mouse model of AD (3xTg-AD) that shows progression of pathology as a function of age versus age-matched wild-type mice. Moreover, we evaluated regional post-synaptic actin polymerization using phalloidin labeling in hippocampal slices. Young (3month-old) 3xTg-AD male mice hippocampus exhibited decreased GluN2B Tyr1472 phosphorylation and reduced Src activity. In the cortex, decreased Src activity correlated with reduced levels of reelin and Dab1, implicating changes in the reelin pathway. We also observed diminished phosphorylated Dab1 and cortactin protein levels in the hippocampus and cortex of young 3xTg-AD male mice. Concordantly with the recognized role of these proteins in actin stabilization, we detected a significant decrease in post-synaptic F-actin in 3month-old 3xTg-AD male CA1 and CA3 hippocampal regions. These data suggest deregulated Src-dependent signaling pathways involving GluN2B-composed NMDARs and post-synaptic actin cytoskeleton depolymerization in the hippocampus in early stages of AD.

Increased Hippocampal Excitability in the 3xTgAD Mouse Model for Alzheimer's Disease In Vivo

Mouse Alzheimer's disease (AD) models develop age- and region-specific pathology throughout the hippocampal formation. One recently established pathological correlate is an increase in hippocampal excitability in vivo. Hippocampal pathology also produces episodic memory decline in human AD and we have shown a similar episodic deficit in 3xTg AD model mice aged 3–6 months. Here, we tested whether hippocampal synaptic dysfunction accompanies this cognitive deficit by probing dorsal CA1 and DG synaptic responses in anaesthetized, 4–6 month-old 3xTgAD mice. As our previous reports highlighted a decline in episodic performance in aged control mice, we included aged cohorts for comparison. CA1 and DG responses to low-frequency perforant path stimulation were comparable between 3xTgAD and controls at both age ranges. As expected, DG recordings in controls showed paired-pulse depression; however, paired-pulse facilitation was observed in DG and CA1 of young and old 3xTgAD mice. During stimulus trains both short-latency (presumably monosynaptic: ‘direct’) and long-latency (presumably polysynaptic: ‘re-entrant’) responses were observed. Facilitation of direct responses was modest in 3xTgAD animals. However, re-entrant responses in DG and CA1 of young 3xTgAD mice developed earlier in the stimulus train and with larger amplitude when compared to controls. Old mice showed less DG paired-pulse depression and no evidence for re-entrance. In summary, DG and CA1 responses to low-frequency stimulation in all groups were comparable, suggesting no loss of synaptic connectivity in 3xTgAD mice. However, higher-frequency activation revealed complex change in synaptic excitability in DG and CA1 of 3xTgAD mice. In particular, short-term plasticity in DG and CA1 was facilitated in 3xTgAD mice, most evidently in younger animals. In addition, re-entrance was facilitated in young 3xTgAD mice. Overall, these data suggest that the episodic-like memory deficit in 3xTgAD mice could be due to the development of an abnormal hyper-excitable state in the hippocampal formation.

Inmunosenescencia prematura en ratones triple-transgénicos para la enfermedad del Alzheimer

IntroductionA deterioration of the neuroimmunoendocrine network has been observed in Alzheimer's disease (AD). However, the peripheral immune response has hardly been investigated in this pathology. Since some immune function parameters have been established as good markers of the rate of ageing, and can predict longevity, the aim of the present work was to study some of these functions in splenic leucocytes in transgenic mice for AD of different ages. Material and methodsYoung female (4±1 months), adult (9±1 months), and mature (12±1 months) triple-transgenic mice for AD (3xTgAD) and non-transgenic (NTg) control mice of the same ages were used. The chemotaxis, the anti-tumour activity of «natural killer» (NK) cells and the lymphoproliferative response in the presence of the mitogens concanavalin A and lipopolysaccharide, functions that decrease with age, were determined in splenic leucocytes. In addition, the differences in lifespan between 3xTgAD and NTg were studied in parallel using other animals, until their death through natural causes. ResultsIn 3xTgAD, with respect to NTg, chemotaxis decreased at all ages studied, whereas in lymphoproliferative response this reduction was shown at 4 months and 9 months. NK activity was diminished only in young 3xTgAD with respect to NTg. The 3xTgAD showed a shorter lifespan than the NTg control group. ConclusionsThe 3xTgAD mice show a premature immunosenescence, which could explain their early mortality. The determination of these immune functions at peripheral level could serve as a marker of the progression of the Alzheimer's disease.