Mutant Human APP Research Articles

Partial inhibition of complex I improves bioenergetics via improved glucose uptake/utilization and mitochondrial function in a cellular model of Alzheimer’s disease

AbstractBackgroundAlzheimer Disease (AD) currently has no effective treatments. A consistent failure of recent clinical trials focused on Aβ or pTau necessitates the identification of novel therapeutic targets and strategies. Since abnormal brain energy homeostasis and mitochondria dysfunction underlie early stages of AD, the development of treatments to restore these defects could be beneficial. We synthesized small molecules that selectively and mildly inhibit mitochondrial complex I (MCI) which restored synaptic and cognitive function in APP/PS1 and 3xTgAD mice. One of the most important mechanisms improved by the treatment included restoration of glucose uptake/utilization that resulted in augmented cellular energetics in mouse brains. However, mechanistic relationship between MCI inhibition, glucose uptake/utilization and mitochondrial function remains to be determined.MethodNeuroblastoma SH‐SY5Y cells that express mutant human APP protein (APPswe) and WT cells were treated with MCI inhibitors in a time‐ and dose‐dependent manner. Flow cytometry was used to measure the translocation of glucose transporters to the cell surface. Changes in protein expression in response to treatment were assessed using Western blot analysis. Changes in mitochondrial morphology were monitored using electron microscopy. Cellular energetics was assessed using a Seahorse Extracellular Flux Analyzer. The non‐radioactive Glucose Uptake‐Glo™ assay was utilized for measuring glucose uptake in cells.ResultsTreatment of with SH‐SY5Y‐APPswe cells with MCI inhibitors within an hour increased surface translocation of GLUTs enhancing glucose uptake and utilization. The effect persisted for 48 hrs. The mechanism of enhanced glucose uptake was associated with AMPK‐dependent activation of NRF2 pathway but not AKT. Furthermore, the improvement in the bioenergetic parameters including Spare Respiratory Capacity, an indicator of the mitochondrial ability to produce energy under stress conditions, and ATP levels were found 24 hrs after the compound treatment. Treatment activated mitochondrial morphofunctional pathway (e.g., fission, fusion, biogenesis and turnover) consistent with the improved cellular bioenergetics. Similar mechanisms were confirmed in the brain tissue of 3xTgAD mice treated with one of the compounds.ConclusionsPartial MCI inhibition activates multiple mechanisms essential for improved energy homeostasis and mitochondrial function. These mechanisms could be consistently induced by our compounds in vivo and in human brain cells support translational potential of this therapeutic strategy.

The AppNLGF mouse model of amyloid pathology: age‐related behavioural characterisation

AbstractBackground2nd generation knock‐in models of amyloid pathology have been generated to overcome the non‐specific effects of the overexpression of human APP mutation in mice (Saito et al., 2014). AppNLGF mice are a relatively new model that expresses human mutant APP at endogenous levels without cellular artefacts noted in transgenic mice (Sasaguri et al., 2022). The key question of how this more restricted expression of human APP mutations influences behaviour in mice has yet to be fully understood. The aim of this study was to establish the profile of memory and anxiety processes in AppNLGF mice with age.MethodsTo monitor the effect of progressive Aβ deposition, AppNLGF mice underwent testing at 4‐6, 8‐10, 12‐14, and 22‐23 months old. Behavioural tests were selected to interrogate key neuronal networks affected in AD. Spatial recognition memory was assessed using the Object Location Test (OLT). Spatial working memory was tested using Spontaneous Alternation (SA) testing. Anxiety‐like behaviours were observed with Open Field (OF) and Elevated Plus Maze (EPM) tests.ResultsDuring OLT, AppNLGF mice showed no significant difference in object discrimination compared to control animals at all ages tested. However, at 8‐10 and 22‐23m/o, AppNLGFs were unable to discriminate the object in the novel location. Furthermore, AppNLGFs showed intact alternation in SA testing until 22‐23m/o. Interestingly, AppNLGFs exhibited decreased inner zone exploration in OF testing at 22‐23m/o, but consistently showed substantial preference for open arms in the EPM test from as early as 4m/o.ConclusionThe AppNLGF model shows subtle cognitive deficits that are most apparent at an advanced age. The severity of the deficits is relatively mild compared to transgenic models assessed on similar tasks. This is probably related to the absence of overexpression‐related artefacts and may speak to the more subtle effects of amyloid on synaptic plasticity. These mice also lack key cellular AD hallmarks such as tau hyperphosphorylation and cell loss. Nevertheless, AppNLGFs robustly display substantial early anxiolytic‐like behaviour in the EPM test, and anxiogenic‐like behaviour in the OF test at an advanced age, suggesting that amyloid pathology may contribute to behavioural and neuropsychiatric changes.

INPP5D modulates TREM2 loss‐of‐function phenotypes in a β‐amyloidosis mouse model

AbstractBackgroundThe genetic associations of TREM2 loss‐of‐function variants with Alzheimer disease (AD) indicate the protective roles of microglia in AD pathogenesis. Functional deficiencies of TREM2 disrupt microglial clustering around amyloid β (Aβ) plaques, impair their transcriptional response to Aβ, and worsen neuritic dystrophy. However, the molecular mechanism underlying these phenotypes remains unclear.MethodsIn this study, we investigated the pathological role of another AD risk gene, INPP5D, encoding a phosphoinositide PI(3,4,5)P3 phosphatase expressed in microglia by crossing Tyrobp knockout, Inpp5d knockout and human mutant APP knockin mice.ResultIn a Tyrobp‐deficient TREM2 loss‐of‐function mouse model, Inpp5d haplodeficiency restored the association of microglia with Aβ plaques, partially restored plaque compaction and astrogliosis, and reduced phosphorylated tau+ dystrophic neurites. RNA‐sequencing of isolated microglia demonstrated that the expression of “disease‐associated microglia” genes is not responsible for these beneficial effects.ConclusionOur results suggest that INPP5D acts downstream of TREM2/TYROBP to regulate the microglial barrier against Aβ toxicity, thereby modulates Aβ‐dependent pathological conversion of tau.

E674Q (Shanghai APP mutant), a novel amyloid precursor protein mutation, in familial late-onset Alzheimer's disease

Identified as the pathogenic genes of Alzheimer's disease (AD), APP, PSEN1, and PSEN2 mainly lead to early-onset AD, whose course is more aggressive, and atypical symptoms are more common than sporadic AD. Here, a novel missense mutation, APP E674Q (also named “Shanghai APP”), was detected in a Chinese index patient with typical late-onset AD (LOAD) who developed memory decline in his mid-70s. The results from neuroimaging were consistent with AD, where widespread amyloid β deposition was demonstrated in 18F-florbetapir Positron Emission Tomography (PET). APP E674Q is close to the β-secretase cleavage site and the well-studied Swedish APP mutation (KM670/671NL), which was predicted to be pathogenic in silico. Molecular dynamics simulation indicated that the E674Q mutation resulted in a rearrangement of the interaction mode between APP and BACE1 and that the E674Q mutation was more prone to cleavage by BACE1. The in vitro results suggested that the E674Q mutation was pathogenic by facilitating the BACE1-mediated processing of APP and the production of Aβ. Furthermore, we applied an adeno-associated virus (AAV)-mediated transfer of the human E674Q mutant APP gene to the hippocampi of two-month-old C57Bl/6 J mice. AAV-E674Q-injected mice exhibited impaired learning behavior and increased pathological burden in the brain, implying that the E674Q mutation had a pathogenicity that bore a comparison with the classical Swedish mutation. Collectively, we report a strong amyloidogenic effect of the E674Q substitution in AD. To our knowledge, E674Q is the only pathogenic mutation within the amyloid processing sequence causing LOAD.

The Role of Physical Activity in Inflammation and Neurogenesis in 5xFAD Mice

Objective/Hypothesis: The apoliprotein E epsilon 4 (APOE4) variant is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD) and is associated with accelerated clinical disease. Increasing evidence suggests that the immune system plays a direct role in AD pathogenesis, and several studies show that APOE genotype alter inflammatory pathways. However, the mechanism by which APOE4 influences the immune system is unknown. Physical activity (PA) is thought to play a neuroprotective role in late-onset AD and represents a possible strategy to attenuate cognitive decline and amyloid beta deposition in APOE4 carriers. We hypothesize that PA counteracts the negative inflammatory effects mediated by APOE4 by promoting a protective microglial response.
 Methods: Mice with humanized APOE3 and APOE4 were crossed to 5xFAD transgenic mice, which carry mutant human APP and presenilin genes to drive amyloid pathology and neurodegeneration. The resultant transgenic mice (5xFAD;APOE4/4 and 5xFAD;APOE3/3) were subjected to a voluntary wheel running paradigm or stationary wheels for 4 months. We used immunohistochemical and ELISA-based techniques to evaluate microglia and neurons. The microglia disease-associated phenotype was assessed by Clec7a staining while homeostatic microglia markers were evaluated by P2ry12 staining. Neuronal numbers in cortical lamina V were evaluated by NeuN staining. Neurogenesis was evaluated by doublecortin staining.
 Results: We expect to see an increase in Clec7a and a decrease in P2ry12 staining on microglia surrounding beta amyloid plaques in 5xFAD;APOE4/4 mice subjected to running compared to mice housed with stationary wheels. In addition, we anticipate PA will ameliorate neuronal loss and promote neurogenesis in the subgranular zone of the dentate gyrus in the hippocampus.
 Conclusion/Potential Impact: The study will support the benefit of PA in attenuating the progression of late-onset AD in individuals with the APOE4 risk allele. The study will also elucidate the relationship between microglial responses, APOE genotype, and PA in AD.

Neurovascular Uncoupling in TgF344‐AD Rats is Associated with Reduced Cerebral Capillary Endothelial Kir2.1 Expression

AbstractBackgroundAlzheimer’s disease (AD) is an emerging global health care crisis. Although reduced cerebral blood flow (CBF) is an early and persistent symptom in developing cognitive deficits, the underlying molecular and cellular mechanisms remain unclear. Recent studies suggested that restoration of capillary endothelial cells (ECs) inward‐rectifier potassium channels (Kir2.1) currents reversed cerebral hypoperfusion and enhanced neurovascular coupling in AD mice. The present study aims to investigate whether capillary ECs Kir2.1 expression is reduced in TgF344‐AD rats and whether it contributes to neurovascular uncoupling and cognitive deficits.MethodThree to six months old TgF‐344 AD rats expressing mutant human APP and PS1 and their age‐matched wildtype (WT) rats were studied. An eight‐arm water maze was used to test spatial learning and short and long‐term memory. Brain perfusion was mapped with a laser speckle imaging system. Whisker stimulation‐induced functional hyperemia was measured using laser Doppler flowmetry in vivo. Brain Kir2.1 expression was compared using western blot. Cerebral parenchymal arterioles (PAs) with attached capillaries were freshly isolated from AD and WT rats and mounted in a pressure myography. PAs response to capillaries focally injected KCL (10 mM) and Kir2.1 inhibitor (ML133) was compared.ResultAD rats exhibited learning and memory dysfunction at 6‐month of age compared with WT controls. However, cerebral hypoperfusion was observed in AD rats starting from 4‐month of age. Similarly, impaired functional hyperemia in AD rats started at 4‐month of age, which was exacerbated in 6‐month old AD rats. The expression of Kir2.1 in 6‐month AD brain tissue was significantly lower than in WT rats. AD PAs displayed less vasodilation (110.96 ± 3.92 % vs. 122.93 ± 1.97 %) and vasoconstriction (92.16 ± 3.47 % vs. 78.04 ± 2.96 %) in response to capillary administered KCL and ML133, respectively, compared to WT vessels.ConclusionReduced brain hypoperfusion precedes cognitive impairment in AD rats, associated with attenuated capillary EC Kir2.1 expression and neurovascular uncoupling.

Granulocyte‐Macrophage Colony‐Stimulating Factor Reduces Two Major Pathological Hallmarks of Alzheimer’s Disease and Astrogliosis in the TgF344‐AD Rat Model

AbstractBackgroundGranulocyte‐macrophage colony‐stimulating factor (GM‐CSF), a hematopoietic growth factor and the innate immune system modulator, has shown therapeutic and neuroprotective effects in animal models of Alzheimer’s disease (AD), Parkinson’s disease, Down syndrome, stroke, and normal aging. Recently, Potter et al. (2021) published the results of a Phase2 clinical trial in mild‐to‐moderate AD subjects showing that recombinant human GM‐CSF (sargramostim) treatment shifted both MMSE scores and plasma biomarkers of neurodegeneration toward normal. Cohen et al. (2013) developed a transgenic rat model (TgF344‐AD) that expresses the Swedish mutant human APP (APPsw) and mutant human presenilin 1 (PSEN1 deltaE9) genes that cause familial AD. In addition to cerebral amyloidosis, neuronal loss, and cognitive deficits, TgF344‐AD rats also show age‐dependent tauopathy including overexpression of phosphorylated tau and neurofibrillary tangle formation, which are not present in mouse models of AD. To evaluate the potential ability of GM‐CSF to reverse and/or prevent the full range of AD pathology, we treated ∼20‐month‐old TgF344‐AD rats with GM‐CSF or saline, and investigated AD pathology in the brain and retina. We also analyzed biomarkers in plasma using MSD and SIMOA neuroinflammation panels.MethodTgF344‐AD rats were injected subcutaneously with GM‐CSF (83.3ug/kg/day; 5days/week) or with saline (200ul/day) for 24 injections total over 32 days. On day 32, blood samples were collected, plasma samples were used for biomarkers assays, and brain and retinal tissues were processed for immunohistochemistry and immunoblotting.ResultTgF344‐AD rats treated with GM‐CSF showed a significant reduction in amyloid plaque deposition in the cortex and a strong trend towards reduced plaque deposition in the hippocampal CA region compared to saline‐treated TgF344‐AD rats. We also observed a trend of reduced phosphorylated tau expression in the cortex and hippocampal region in GM‐CSF‐treated TgF344‐AD rats. In addition, GM‐CSF treatment significantly reduced astrogliosis in the CA1 and dentate gyrus/hilus regions of TgF344‐AD rats.ConclusionAlthough experiments are ongoing, our findings indicate that GM‐CSF treatment rescues two major pathological hallmarks of AD and neuroinflammation (astrogliosis) in TgF344‐AD rats. Our results suggest that GM‐CSF/sargramostim/Leukine® may be a potential therapeutic not only for reducing amyloidosis, but also for reducing levels of hyperphosphorylated tau and other relevant pathological manifestations of AD.

Intranasal interferon-beta alleviates anxiety and depressive-like behaviors by modulating microglia polarization in an Alzheimer's disease model

Alzheimer's disease (AD) patients frequently experience neuropsychiatric symptoms (NPS), which are linked to a lower quality of life and a faster rate of disease progression. A growing body of research indicates that several microglial phenotypes control the inflammatory response and are crucial in the pathophysiology of AD-related NPS. Given the crucial role played by inflammatory mediators produced by microglia in developing of NPS, interferon-beta (IFNβ), a cytokine with anti-inflammatory capabilities, maybe a successful treatment for NPS caused by AD. In this investigation, using a rat model of AD, we examined the impact of intranasal treatment of IFNβ on anxious/depressive-like behavior and microglial M1/M2 polarization. The rat hippocampus was bilaterally injected with lentiviruses harboring mutant human amyloid precursor protein. Rats were given recombinant IFNβ1a (68,000 IU/rat) via the intranasal route, starting on day 23 following viral infection and continuing until day 49. On days 47–49, the elevated plus maze, forced swim, and tail suspension tests were applied to measure anxiety- and depressive-like behavior. Additionally, qPCR was utilized to quantify the expression of M1 markers (CD68, CD86, and CD40) and M2 markers (Ym1, CD206, Arg1, GDNF, BDNF, and SOCS1). Our findings demonstrated that decreased M2 marker expression is accompanied by anxious/depressive-like behavior when the mutant human APP gene is overexpressed in the hippocampus. In the rat model of AD, IFNβ therapy reduces anxious/depressive-like behaviors, at least in part by polarizing microglia towards M2. Therefore, IFNβ may be a viable therapeutic drug for reducing NPS in the context of AD.

Effects of Deferasirox in Alzheimer's Disease and Tauopathy Animal Models.

The accumulation of iron may contribute to Alzheimer’s disease (AD) and other tauopathies. The iron chelator desferrioxamine slows disease progression in AD patients. However, desferrioxamine requires injection, which is inconvenient and may hinder compliance. We therefore tested an oral iron chelator, desferasirox (Exjade), in transgenic animal models. Tg2576 mice overexpress the mutant human APP protein and produce the Aβ peptide. JNPL3 mice (Tau/Tau) overexpress the mutant human tau protein. Crossing these produced APP/Tau mice, overexpressing both APP and tau. Treating the three models with 1.6 mg deferasirox thrice weekly from age 8 to 14 months did not affect memory as measured by contextual fear conditioning or motor function as measured by rotarod, but tended to decrease hyperphosphorylated tau as measured by AT8 immunohistochemistry and immunoblotting. Deferasirox might act by decreasing iron, which aggregates tau, or directly binding tau to inhibit aggregation.

Granulocyte-macrophage colony-stimulating factor reverses Alzheimer's disease pathology in the tgf344-AD rat model.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, has shown therapeutic and neuroprotective effects in animal models of Alzheimer's disease (AD), Parkinson's disease, Down syndrome, and stroke. In a retrospective study, we reported that recombinant human GM-CSF (sargramostim) treatment led to improved cognition in patients with cognitive impairment due to chemotherapy. Our recently completed Phase 2 clinical trial in AD showed that sargramostim treatment increased toward normal both MMSE scores and plasma biomarkers of neurodegeneration. Cohen et al. (2013) developed a transgenic rat model (TgF344-AD) that expresses the Swedish mutant human APP (APPsw) and mutant human presenilin 1 (PSEN1) genes that cause familial AD. Interestingly, in addition to cerebral amyloidosis, neuronal loss, and cognitive deficits, TgF344-AD rats also show age-dependent tauopathy including overexpression of phosphorylated tau and neurofibrillary tangle formation, which do not arise in mouse models of AD. To evaluate the potential ability of GM-CSF to reverse and/or prevent the full range of AD pathology, we treated ∼20-month-old TgF344-AD rats with GM-CSF or saline, and investigated AD pathology in the brain and retina. We also analyzed biomarkers in plasma using Meso Scale Discovery (MSD) and SIMOA neuroinflammation panels. TgF344-AD rats were injected subcutaneously with GM-CSF (83.3 ug/kg/day; 5 days/week) or with saline (200 ul/day) for 24 injections total over 32 days. On day 32, blood samples were collected, plasma samples were isolated, and brain and retina tissues were processed for immunohistochemistry and immunoblotting. TgF344-AD rats treated with GM-CSF showed a significant reduction in amyloid plaque deposition in the cortex compared to saline-treated TgF344-AD rats. We also observed a trend of reduced amyloid plaques in the CA region of the hippocampus (p=0.06) in GM-CSF-treated TgF344-AD rats. In ongoing experiments, we are investigating the effects of GM-CSF treatment on tauopathy and neuroinflammation in the brain and retina and on plasma biomarkers. Although experiments are in progress, our preliminary findings indicate that GM-CSF treatment rescues some AD pathology in TgF344-AD rats. Complete results will determine the therapeutic potential for GM-CSF/sargramostim/Leukine® not only for reducing amyloidosis, but also on other relevant pathological manifestations of AD.

T1AM-TAAR1 signalling protects against OGD-induced synaptic dysfunction in the entorhinal cortex

Abnormalities in thyroid hormones (TH) availability and/or metabolism have been hypothesized to contribute to Alzheimer's disease (AD) and to be a risk factor for stroke. Recently, 3-iodothyronamine (T1AM), an endogenous amine putatively derived from TH metabolism, gained interest for its ability to promote learning and memory in the mouse. Moreover, T1AM has been demonstrated to rescue the β-Amyloid dependent LTP impairment in the entorhinal cortex (EC), a brain area crucially involved in learning and memory and early affected during AD. In the present work, we have investigated the effect of T1AM on ischemia-induced EC synaptic dysfunction. In EC brain slices exposed to oxygen-glucose deprivation (OGD), we demonstrated that the acute perfusion of T1AM (5 μM) was capable of preventing ischemia-induced synaptic depression and that this protective effect was mediated by the trace amine-associated receptor 1 (TAAR1). Moreover, we demonstrated that activation of the BDNF-TrkB signalling is required for T1AM action during ischemia. The protective effect of T1AM was more evident when using EC slices from transgenic mutant human APP (mhAPP mice) that are more vulnerable to the effect of OGD. Our results confirm that the TH derivative T1AM can rescue synaptic function after transient ischemia, an effect that was also observed in a Aβ-enriched environment.

Leucine Carboxyl Methyltransferase 1 Overexpression Protects Against Cognitive and Electrophysiological Impairments in Tg2576 APP Transgenic Mice.

Background:The serine/threonine protein phosphatase, PP2A, is thought to play a central role in the molecular pathogenesis of Alzheimer’s disease (AD), and the activity and substrate specificity of PP2A is regulated, in part, through methylation and demethylation of its catalytic subunit. Previously, we found that transgenic overexpression of the PP2A methyltransferase, LCMT-1, or the PP2A methylesterase, PME-1, altered the sensitivity of mice to impairments caused by acute exposure to synthetic oligomeric amyloid-β (Aβ).Objective:Here we sought to test the possibility that these molecules also controlled sensitivity to impairments caused by chronically elevated levels of Aβ produced in vivo.Methods:To do this, we examined the effects of transgenic LCMT-1, or PME-1 overexpression on cognitive and electrophysiological impairments caused by chronic overexpression of mutant human APP in Tg2576 mice.Results:We found that LCMT-1 overexpression prevented impairments in short-term spatial memory and synaptic plasticity in Tg2576 mice, without altering APP expression or soluble Aβ levels. While the magnitude of the effects of PME-1 overexpression in Tg2576 mice was small and potentially confounded by the emergence of non-cognitive impairments, Tg2576 mice that overexpressed PME-1 showed a trend toward earlier onset and/or increased severity of cognitive and electrophysiological impairments.Conclusion:These data suggest that the PP2A methyltransferase, LCMT-1, and the PP2A methylesterase, PME-1, may participate in the molecular pathogenesis of AD by regulating sensitivity to the pathogenic effects of chronically elevated levels of Aβ.

Elevated circulating amyloid concentrations in obesity and diabetes promote vascular dysfunction.

Diabetes, obesity, and Alzheimer’s disease (AD) are associated with vascular complications and impaired nitric oxide (NO) production. Furthermore, increased β-site amyloid precursor protein–cleaving (APP-cleaving) enzyme 1 (BACE1), APP, and β-amyloid (Aβ) are linked with vascular disease development and increased BACE1 and Aβ accompany hyperglycemia and hyperlipidemia. However, the causal relationship between obesity and diabetes, increased Aβ, and vascular dysfunction is unclear. We report that diet-induced obesity (DIO) in mice increased plasma and vascular Aβ42 that correlated with decreased NO bioavailability, endothelial dysfunction, and increased blood pressure. Genetic or pharmacological reduction of BACE1 activity and Aβ42 prevented and reversed, respectively, these outcomes. In contrast, expression of human mutant APP in mice or Aβ42 infusion into control diet–fed mice to mimic obese levels impaired NO production, vascular relaxation, and raised blood pressure. In humans, increased plasma Aβ42 correlated with diabetes and endothelial dysfunction. Mechanistically, higher Aβ42 reduced endothelial NO synthase (eNOS), cyclic GMP (cGMP), and protein kinase G (PKG) activity independently of diet, whereas endothelin-1 was increased by diet and Aβ42. Lowering Aβ42 reversed the DIO deficit in the eNOS/cGMP/PKG pathway and decreased endothelin-1. Our findings suggest that BACE1 inhibitors may have therapeutic value in the treatment of vascular disease associated with diabetes.

THE MOLECULAR TWEEZER CLR01 AMELIORATES PATHOLOGY IN THE P301S-TAU MOUSE MODEL

Tau self-assembly into neurotoxic oligomers and fibrils underlies the pathophysiology in tauopathies. Molecular tweezers (MTs) act as unique, artificial nanochaperones and are the first small molecules whose activity as modulators/inhibitors of abnormal protein self-assembly has been discovered using a rational approach. Previously, we evaluated the effect of a lead MT, called CLR01, in the 3×Tg mouse model, which overexpresses mutant human PS1(M146V), APP(KM670/671NL), and tau(P301L). Immunohistochemistry (IHC) showed robust reduction of amyloid plaques, neurofibrillary tangles, and microgliosis. Because tau hyperphosphorylation is downstream of Aβ insults, the question whether CLR01 affected tau directly or indirectly remained open. Therefore, we asked if CLR01 could be used for treatment of tauopathies in the absence of amyloid pathology and set up to answer this question using the P301S-tau mouse model. The study included five groups of mice, each comprising 8 males and 8 females. The treatment groups included three Tg groups, administered 0, 0.3, or 1.0 mg/kg per day CLR01 and two wild-type littermate groups receiving 0 or 1.0 mg/kg per day CLR01. CLR01 was administered for 35 days via osmotic minipumps implanted subcutaneously at 6−6.5 months of age. The mice were weighed out and grip-strength and open field tests were performed in the beginning, middle (grip-strength only), and end of the treatment. No adverse effects were observed in any of the groups. CLR01 prevented a decline in grip-strength observed in vehicle-treated Tg mice. The mice did not show deficits in any open-field test precluding evaluation of drug effect. Following completion of the treatment, the mice were sacrificed and the brains were collected and divided into the two hemispheres. One hemisphere was fixed and sectioned for IHC analysis and the other hemisphere was dissected into regions of interest. Brain regions were snap-frozen and extracted into soluble, membrane-bound, and insoluble fractions. ELISA results and cell culture-based seeding assay show that CLR01 treatment reduced tau hyperphosphorylation and formation of tau seeds. Analysis of tau oligomerization is also underway. Our initial results strongly suggest CLR01's target engagement and beneficial therapeutic effect.

Amyloid pathology enhances seeding and propagation of fibrillar endogenous non-mutant tau in vivo

Amyloid pathology enhances seeding and propagation of fibrillar endogenous non-mutant tau in vivo

Amyloid β Is Not the Major Factor Accounting for Impaired Adult Hippocampal Neurogenesis in Mice Overexpressing Amyloid Precursor Protein.

SummaryAdult hippocampal neurogenesis was impaired in several Alzheimer's disease models overexpressing mutant human amyloid precursor protein (hAPP). However, the effects of wild-type hAPP on adult neurogenesis and whether the impaired adult hippocampal neurogenesis was caused by amyloid β (Aβ) or APP remained unclear. Here, we found that neurogenesis was impaired in the dentate gyrus (DG) of adult mice overexpressing wild-type hAPP (hAPP-I5) compared with controls. However, the adult hippocampal neurogenesis was more severely impaired in hAPP-I5 than that in hAPP-J20 mice, which express similar levels of hAPP mRNA but much higher levels of Aβ. Furthermore, reducing Aβ levels did not affect the number of doublecortin-positive cells in the DG of hAPP-J20 mice. Our results suggested that hAPP was more likely an important factor inhibiting adult neurogenesis, and Aβ was not the major factor affecting neurogenesis in the adult hippocampus of hAPP mice.

Preclinical Assessment of Young Blood Plasma for Alzheimer Disease

Alzheimer disease (AD) pathology starts long before clinical symptoms manifest, and there is no therapy to treat, delay, or prevent the disease. A shared blood circulation between 2 mice (aka parabiosis) or repeated injections of young blood plasma (plasma from 2- to 3-month-old mice) into old mice has revealed benefits of young plasma on synaptic function and behavior. However, to our knowledge, the potential benefit of young blood has not been tested in preclinical models of neurodegeneration or AD. To determine whether young blood plasma ameliorates pathology and cognition in a mouse model for AD and could be a possible future treatment for the disease. In this preclinical study, mice that harbor a human mutant APP gene, which causes familial AD, were aged to develop AD-like disease including accumulation of amyloid plaques, loss of synaptic and neuronal proteins, and behavioral deficits. The initial parabiosis studies were done in 2010, and the final studies were conducted in 2014. Alzheimer disease model mice were then treated either by surgically connecting them with a young healthy mouse, thus providing a shared blood circulation through parabiosis, or through repeated injections of plasma from young mice. Neuropathological parameters and changes in hippocampal gene expression in response to the treatment were assessed. In addition, cognition was tested in AD model mice intravenously injected with young blood plasma. Aged mutant amyloid precursor protein mice with established disease showed a near complete restoration in levels of synaptic and neuronal proteins after exposure to young blood in parabiosis (synaptophysin P = .02; calbindin P = .02) or following intravenous plasma administration (synaptophysin P < .001; calbindin P = .14). Amyloid plaques were not affected, but the beneficial effects in neurons in the hippocampus were accompanied by a reversal of abnormal extracellular receptor kinase signaling (P = .05), a kinase implicated in AD. Moreover, young plasma administration was associated with improved working memory (P = .01) and associative memory (P = .02) in amyloid precursor protein mice. Factors in young blood have the potential to ameliorate disease in a model of AD.

The Tau/A152T mutation, a risk factor for frontotemporal-spectrum disorders, leads to NR2B receptor-mediated excitotoxicity.

We report on a novel transgenic mouse model expressing human full-length Tau with the Tau mutation A152T (hTau(AT)), a risk factor for FTD-spectrum disorders including PSP and CBD Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis-sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short- or long-term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage-gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau(AT) causes excitotoxicity mediated by NR2B-containing NMDA receptors due to enhanced extracellular glutamate.

Reserpine Improves Working Memory

Despite exhaustive search, no drug is in sight for AD. Earlier, we reported that reserpine, an antihypertensive and antipsychotic drug, ameliorates Amyloid beta (Aβ-AD causing peptide) toxicity and confers several positive enhancements in the C. elegans model system. Here, we evaluate whether reserpine can provide protection against working memory and against AD in the mouse model. Reserpine (0.08 mg) was administered orally on alternate days to the non-Tg and accelerated Aβ deposition (at 2 months of age)and cognitive deficit (4 months of age) developing 5XFAD AD Tg mouse model expressing mutant human APP (3 familial mutations) and human Presenilin1(2 familial mutations) in the neurons, and follow their working memory for 2 months using the spontaneous Y-maze alteration behavioral paradigm. Reserpine enhanced working memory in non-Tg mice and improved the cognitive deficit in the 5XFAD AD Tg mice. Hence, reserpine can be considered for a detailed evaluation in the 3X Tg AD mouse model and a pilot study in AD patients.

APP overexpression in the absence of NPC1 exacerbates metabolism of amyloidogenic proteins of Alzheimer's disease.

Amyloid-β (Aβ) peptides originating from β-amyloid precursor protein (APP) are critical in Alzheimer's disease (AD). Cellular cholesterol levels/distribution can regulate production and clearance of Aβ peptides, albeit with contradictory outcomes. To better understand the relationship between cholesterol homeostasis and APP/Aβ metabolism, we have recently generated a bigenic ANPC mouse line overexpressing mutant human APP in the absence of Niemann-Pick type C-1 protein required for intracellular cholesterol transport. Using this unique bigenic ANPC mice and complementary stable N2a cells, we have examined the functional consequences of cellular cholesterol sequestration in the endosomal-lysosomal system, a major site of Aβ production, on APP/Aβ metabolism and its relation to neuronal viability. Levels of APP C-terminal fragments (α-CTF/β-CTF) and Aβ peptides, but not APP mRNA/protein or soluble APPα/APPβ, were increased in ANPC mouse brains and N2a-ANPC cells. These changes were accompanied by reduced clearance of peptides and an increased level/activity of γ-secretase, suggesting that accumulation of APP-CTFs is due to decreased turnover, whereas increased Aβ levels may result from a combination of increased production and decreased turnover. APP-CTFs and Aβ peptides were localized primarily in early-/late-endosomes and to some extent in lysosomes/autophagosomes. Cholesterol sequestration impaired endocytic-autophagic-lysosomal, but not proteasomal, clearance of APP-CTFs/Aβ peptides. Moreover, markers of oxidative stress were increased in vulnerable brain regions of ANPC mice and enhanced β-CTF/Aβ levels increased susceptibility of N2a-ANPC cells to H2O2-induced toxicity. Collectively, our results show that cellular cholesterol sequestration plays a key role in APP/Aβ metabolism and increasing neuronal vulnerability to oxidative stress in AD-related pathology.