Neuropathology of Mice Carrying Mutant APPswe and/or PS1M146L Transgenes: Alterations in the p75 Cholinergic Basal Forebrain Septohippocampal Pathway

Cholinergic basal forebrain (CBF) projection systems are defective in late Alzheimer's disease (AD). We examined the brains of 12-month-old singly and doubly transgenic mice overexpressing mutant amyloid precursor protein (APP_swe) and/or presenilin-1 (PS1_M146L) to investigate the effects of these AD-related genes on plaque and tangle pathology, astrocytic expression, and the CBF projection system. Two types of β-amyloid (Aβ)-immunoreactive (ir) plaques were observed: type 1 were darkly stained oval and elongated deposits of Aβ, and type 2 were diffuse plaques containing amyloid fibrils. APP_swe and PS1_M146L mouse brains contained some type 1 plaques, while the doubly transgenic (APP_swe/PS1_M146L) mice displayed a greater abundance of types 1 and 2 plaques. Sections immunostained for the p75 NGF receptor (p75^NTR) revealed circular patches scattered throughout the cortex and hippocampus of the APP_swe/PS1_M146L mice that contained Aβ, were innervated by p75^NTR-ir neurites, but displayed virtually no immunopositive neurons. Tau pathology was not seen in any transgenic genotype, although a massive glial response occurred in the APP_swe/PS1_M146L mice associated with amyloid plaques. Stereology revealed a significant increase in p75^NTR-ir medial septal neurons in the APP_swe and PS1_M146L singly transgenic mice compared to the APP_swe/PS1_M146L mice. No differences in size or optical density of p75^NTR-ir neurons were observed in these three mutants. p75^NTR-ir fibers in hippocampus and cortex were more pronounced in the APP_swe and PS1_M146L mice, while the APP_swe/PS1_M146L mice showed the least p75^NTR-ir fiber staining. These findings suggest a neurotrophic role for mutant APP and PS1 upon cholinergic hippocampal projection neurons at 12 months of age.     

Deficiency in the inner mitochondrial membrane peptidase 2-like (Immp21) gene increases ischemic brain damage and impairs mitochondrial function

Mitochondrial dysfunction plays an important role in mediating ischemic brain damage. Immp2l is an inner mitochondrial membrane peptidase that processes mitochondrial protein cytochrome c1 (Cyc1). Homozygous mutation of Immp2l (Immp2l^{Tg(Tyr)979Ove} or Immp2l^−/−) elevates mitochondrial membrane potential, increases superoxide (O₂⁻) production in the brain and impairs fertility. The objectives of this study are to explore the effects of heterozygous mutation of Immp2l (Immp2l^+/−) on ischemic outcome and to determine the influence of Immp2l deficiency on brain mitochondria after stroke. Male Immp2l^+/− and wild-type (WT) mice were subjected to 1-h focal cerebral ischemia. Their brains were harvested after 5 and 24-h of reperfusion. The results showed that infarct volume and DNA oxidative damage significantly increased in the Immp2l^+/− mice. There were no obvious cerebral vasculature abnormalities between the two types of mice viewed by Indian ink perfusion. The increased damage in Immp2l^+/− mice was associated with early increase in O₂⁻ production. Mitochondrial respiratory rate, total mitochondrial respiratory capacity and mitochondrial respiratory complex activities were decreased at 5-h of recirculation in Immp2l^+/− mice compared to WT mice. Our results suggest that Immp2l deficiency increases ischemic brain damage by enhancing O₂⁻ production and damaging mitochondrial functional performance.     

Analysis of CX3CR1 haplodeficiency in male and female APPswe/PSEN1dE9 mice along Alzheimer disease progression

Microglia, the resident immune cells of the brain, have recently emerged as key players in Alzheimer Disease (AD) pathogenesis, but their roles in AD remain largely elusive and require further investigation. Microglia functions are readily altered when isolated from their brain environment, and microglia reporter mice thus represent valuable tools to study the contribution of these cells to neurodegenerative diseases such as AD. The CX3CR1^+/eGFP mice is one of the most popular microglia reporter mice, and has been used in numerous studies to investigate in vivo microglial functions, including in the context of AD research. However, until now, the impact of CX3CR1 haplodeficiency on the typical features of Alzheimer Disease has not been studied in depth.To fill this gap, we generated APP^swe/PSEN1^dE9:CX3CR1^+/eGFP mice and analyzed these mice for Alzheimer’s like pathology and neuroinflammation hallmarks. More specifically, using robust multifactorial statistical and multivariate analyses, we investigated the impact of CX3CR1 deficiency in both males and females, at three typical stages of the pathology progression: at early stage when Amyloid-β (Aβ) deposition just starts, at intermediate stage during Aβ accumulation phase and at more advanced stages when Aβ plaque number stabilizes. We found that CX3CR1 haplodeficiency had little impact on the progression of the pathology in the APP^swe/PSEN1^dE9 model and demonstrated that the APP^swe/PSEN1^dE9:CX3CR1^+/eGFP line is a relevant and useful model to study the role of microglia in Alzheimer Disease. In addition, although Aβ plaques density is higher in females compared to age-matched males, we show that their glial reaction, inflammation status and memory deficits are not different.     

DHA and cholesterol containing diets influence Alzheimer-like pathology, cognition and cerebral vasculature in APPswe/PS1dE9 mice

Cholesterol and docosahexenoic acid (DHA) may affect degenerative processes in Alzheimer's Disease (AD) by influencing Aβ metabolism indirectly via the vasculature. We investigated whether DHA-enriched diets or cholesterol-containing Typical Western Diets (TWD) alter behavior and cognition, cerebral hemodynamics (relative cerebral blood volume (rCBV)) and Aβ deposition in 8- and 15-month-old APP_swe/PS1_dE9 mice. In addition we investigated whether changes in rCBV precede changes in Aβ deposition or vice versa. Mice were fed regular rodent chow, a TWD-, or a DHA-containing diet. Behavior, learning and memory were investigated, and rCBV was measured using contrast-enhanced MRI. The Aβ load was visualized immunohistochemically. We demonstrate that DHA altered rCBV in 8-month-old APP/PS1 and wild type mice[AU1]. In 15-month-old APP/PS1 mice DHA supplementation improved spatial memory, decreased Aβ deposition and slightly increased rCBV, indicating that a DHA-enriched diet can diminish AD-like pathology. In contrast, TWD diets decreased rCBV in 15-month-old mice. The present data indicate that long-term dietary interventions change AD-like pathology in APP/PS1 mice. Additionally, effects of the tested diets on vascular parameters were observed before effects on Aβ load were noted. These data underline the importance of vascular factors in the APP/PS1 mouse model of AD pathology.     

Spatial learning and memory impairment and increased locomotion in a transgenic amyloid precursor protein mouse model of Alzheimer's disease

A well-known example for gene x environment interactions in psychiatry is the one involving the low activity (s) allelic variant of the serotonin transporter (5-HTT) promoter polymorphism (5-HTTLPR) that in the context of stress increases risk for depression. In analogy, 5-HTT knockout rodents are highly responsive to early life, but also adult external stressors, albeit conflicting data have been obtained. In our study on emotion and cognition using homozygous 5-HTT knockout (5-HTT^−/−) and wild-type (5-HTT^+/+) rats we have been confronted with animal facility construction, which were associated with severe lifetime stress (noise and vibrations). To assess the impact of construction stress on well-established 5-HTT^−/− rat phenotypes we conducted ad hoc analyses of 5-HTT^−/− and 5-HTT^+/+ rats that grew up before and during the construction. The reproductive capacity of the parents of the experimental 5-HTT^+/− rats was significantly decreased. Further, 5-HTT^−/− anxiety-related phenotypes in the elevated plus maze and social interaction tests were abolished after construction noise exposure, due to increased anxiety in 5-HTT^+/+ rats and decreased anxiety in 5-HTT^−/− rats (social interaction test only). In addition, reversal learning was improved in 5-HTT^+/+ and, to a milder extent, decreased in 5-HTT^−/− rats. Finally, construction stress genotype-independently increased behavioural despair in the forced swim test. In conclusion, severe construction stress induces 5-HTT genotype-dependent ‘for-better-and-for-worse’ effects. These data importantly contribute to the understanding of 5-HTT gene x environment interactions and show the risk of losing genotype effects by construction stress.     

Amyloid-Beta Mediates Homeostatic Synaptic Plasticity

The physiological role of the amyloid-precursor protein (APP) is insufficiently understood. Recent work has implicated APP in the regulation of synaptic plasticity. Substantial evidence exists for a role of APP and its secreted ectodomain APPsα in Hebbian plasticity. Here, we addressed the relevance of APP in homeostatic synaptic plasticity using organotypic tissue cultures prepared from APP^−/− mice of both sexes. In the absence of APP, dentate granule cells failed to strengthen their excitatory synapses homeostatically. Homeostatic plasticity is rescued by amyloid-β and not by APPsα, and it is neither observed in APP^+/+ tissue treated with β- or γ-secretase inhibitors nor in synaptopodin-deficient cultures lacking the Ca²⁺-dependent molecular machinery of the spine apparatus. Together, these results suggest a role of APP processing via the amyloidogenic pathway in homeostatic synaptic plasticity, representing a function of relevance for brain physiology as well as for brain states associated with increased amyloid-β levels.     

GSK3β is involved in promoting Alzheimer’s disease pathologies following chronic systemic exposure to Porphyromonas gingivalis lipopolysaccharide in amyloid precursor proteinNL-F/NL-F knock-in mice

In line with the strong association between periodontitis and Alzheimer’s disease (AD) clinically, preclinical studies have shown that systemic exposure to Porphyromonas gingivalis (Pg) initiates AD pathologies. However, the involvement of periodontitis in promoting AD pathologies is unclear. In the present study, we provided evidence that chronic systemic exposure to lipopolysaccharide derived from Pg (PgLPS, 1 mg/kg, daily, intraperitoneally) prompted neuroinflammation and tau hyperphosphorylation in 10-month-old of amyloid precursor protein (APP) knock-in mice, a model of AD, carrying the Swedish and Beyreuther/Iberian mutation (APP^NL-F/NL-F). The learning and memory function were assessed using the passive avoidance test. The production of APP, Amyloid (A)β_1-42, cytokines, synaptic proteins and the activation of glycogen synthase kinase (GSK)-3β as well as phosphorylation of tau were analyzed by immunohistochemistry, Western blotting or an enzyme-linked immunosorbent assay (ELISA) in the cortex of APP^NL-F/NL-F mice. We found that systemic exposure of PgLPS for three consecutive weeks induced learning and memory deficits with significantly reduced postsynaptic density protein (PSD95). Increased hyperphosphorylation of tau in multiple residues, including Ser202, Thr231 and Ser396, but not the accumulation of Aβ_1-42 was detected in the neurons of APP^NL-F/NL-F mice. Furthermore, PgLPS increased the GSK3β activity by reducing its phosphorylation of the serine residue at position 9 (Ser9) and promoted neuroinflammation by increasing the expression of interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α) while decreasing that of interleukin-10 (IL-10) and transforming growth factor (TGFβ) in the cortex of APP^NL-F/NL-F mice. Moreover, the PgLPS-increased GSK3β activity was detected in both microglia and neurons, while the PgLPS-increased TNF-α expression was mainly detected in the microglia in the cortex of APP^NL-F/NL-F mice. In in vitro studies, PgLPS (1 µg/ml) stimulation increased the mRNA and protein level of TNF-α in MG6 microglia, which were significantly inhibited by the GSK3β-specific inhibitor TWS119. In contrast, the tau hyperphosphorylation and activation of GSK3β in N2a neurons were enhanced after treatment with conditioned medium from PgLPS-stimulated microglia, which was attenuated after pre-treatment with TNF-α inhibitor. Taken together, these findings indicate that GSK3β is involved in prompting microglia (TNF-α)-dependent tau hyperphosphorylation in neurons, resulting in learning and memory deficits in APP^NL-F/NL-F mice without changes in the Aβ expression during chronic systemic exposure to PgLPS. We propose that dampening GSK3β activation may help delay the periodontitis-promoted pathological progression of AD.     

Mitochondrial dysfunction and accumulation of the β-secretase-cleaved C-terminal fragment of APP in Alzheimer's disease transgenic mice

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-β (Aβ) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the β-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the β-site APP-cleaving enzyme 1 (BACE1^+/−) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6 months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1^+/−·5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1^+/− deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1^+/− deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6 months but not at 12 months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD.     

Role of plasminogen in macrophage accumulation during liver repair

Introduction

Although the involvement of plasminogen in liver repair has been reported, its roles are still poorly understood. Here, we investigated the role of plasminogen in accumulations of macrophages and neutrophils after liver injury in mice with gene deficient of plasminogen (Plg^−/−) or its wild type (Plg^+/+).

Materials and Methods

Mice received traumatic liver injury caused by stabbing on the lobe or hepatic ischemia-reperfusion, and the damaged sites were histologically analyzed.

Results

After the traumatic liver injury, both the stab wound and the damaged tissue were decreased until day 7 in the Plg^+/+ mice. In contrast, both the stab wound and the damaged tissue were still remained until day 7 in the Plg^−/− mice. On day 4 after traumatic liver injury, macrophages were abundant at the surrounding area of the damaged site in the Plg^+/+ mice. However, the macrophage accumulation was impaired in the Plg^−/− mice. After hepatic ischemia-reperfusion injury, macrophage accumulation and decrease in the damaged tissue were also observed in the Plg^+/+ mice until day 7. In contrast, these responses were also impaired in the Plg^−/− mice. Furthermore, neutrophil accumulation at the surrounding area of the damaged site was also impaired in the Plg^−/− mice on day 4 after both liver traumatic liver injury and hepatic ischemia-reperfusion injury.

Conclusions

Our data indicate that plasminogen plays a crucial role in macrophage accumulation together with the neutrophil accumulation after liver injury in both models, which may be essential for triggering the subsequent healing responses including decrease in the damaged tissue.     

Vglut2 haploinsufficiency enhances behavioral sensitivity to MK-801 and amphetamine in mice

Recently developed mouse models have implicated the vesicular glutamate transporter 2 (VGLUT2) in psychostimulant-induced hyperactivity, a behavioral assay that is often applied to evaluate mouse behavior related to positive schizophrenia (SCZ) symptomatology. In present research, we wanted to evaluate further the role of subtle VGLUT2 impairment as a factor underlying SCZ symptomatology. To this end, we evaluated Vglut2 haploinsufficient (Vglut2^+/−) mice and their wildtype littermates in a test battery assessing behaviors related to positive, negative and cognitive SCZ symptom domains. We found in Vglut2^+/− mice an increased locomotor response to amphetamine and an increased sensitivity to the startle-disrupting effects of MK-801, but no impairment in sensorimotor gating. Further on, minor alterations in tests assessing cognitive and negative symptom-related behavior were observed. Possible neurobiological mechanisms of these observations are discussed.     

Overexpression of wild-type human APP in mice causes cognitive deficits and pathological features unrelated to Aβ levels

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPP_wt) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but β-amyloid peptide (Aβ₄₂) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Aβ₄₂ were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Aβ-independent pathogenic pathways in Alzheimer's disease.     

Evaluation of the effects of vitamin A supplementation on adult rat substantia nigra and striatum redox and bioenergetic states: Mitochondrial impairment,increased 3-nitrotyrosine and α-synuclein,but decreased D2 receptor contents

Vitamin A at moderate to high doses is applied in the treatment of some life threatening pathological conditions, for instance cancers. Additionally, vitamin A at low concentrations is a known antioxidant molecule. However, by increasing vitamin A (or its derivatives) concentrations, there is an increase in the levels of oxidative stress markers in several experimental models. Furthermore, it was reported that vitamin A therapy at high doses might induce cognitive decline among the patients, which may become anxious or depressive, for example, depending on vitamin A levels intake. We have previously reported increased levels of oxidative stress markers in rat substantia nigra and striatum. However, the mechanism by which this vitamin altered the redox environment in such rat brain regions remains to be elucidated. In the herein presented work, we have investigated the effects of vitamin A supplementation at clinical doses (1000–9000 IU/kg day^− 1) for 28 days on rat substantia nigra and striatum mitochondrial electron transfer chain (METC) activity, which may produce superoxide anion radical (O₂^−•) when impaired. Additionally, the levels of non-enzymatic antioxidant defenses were evaluated, as well as 3-nitrotyrosine, α- and β-synucleins and TNF-α levels through ELISA assay. We observed impaired METC in both rat brain regions. Moreover, we found increased O₂^−• production and nitrotyrosine content in the nigrostriatal axis of vitamin A-treated rats, suggesting that the use of vitamin A at therapeutic doses may be rethought due to this toxic effects found here.     

Effective components of Chinese herbs reduce central nervous system function decline induced by iron overload

Abnormally increased levels of iron in the brain trigger cascade amplification in Alzheimer’s disease patients, resulting in neuronal death. This study investigated whether components extracted from the Chinese herbs epimedium herb, milkvetch root and kudzuvine root could relieve the abnormal expression of iron metabolism-related protein in Alzheimer’s disease patients. An APP_swe/PS1_ΔE9 double transgenic mouse model of Alzheimer’s disease was used. The intragastric administration of compounds from epimedium herb, milkvetch root and kudzuvine root improved pathological alterations such as neuronal edema, increased the number of neurons, downregulated divalent metal transporter 1 expression, upregulated ferroportin 1 expression, and inhibited iron overload in the cerebral cortex of mice with Alzheimer’s disease. These compounds reduced iron overload-induced impairment of the central nervous system, indicating a new strategy for developing novel drugs for the treatment of Alzheimer’s disease.     

P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation

Introduction

Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology.

Methods and Results

Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and P-selectin-deficient (CD62P^−/−) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet α_IIbβ₃. Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P^−/− mice. In addition, CD62P^−/− mice exhibited thrombosis abnormalities without an α_IIbβ₃ activation defect.

Conclusions

This study demonstrates that the role of platelet P-selectin is not solely adhesive; its binding to PSGL-1 induces platelet activation that enhances platelet aggregation and thrombus formation. Therefore, targeting platelet P-selectin or its ligand PSGL-1 could provide a potential therapeutic approach in the management of thrombotic disorders.     

Deletion of insulin-regulated aminopeptidase in mice decreases susceptibility to pentylenetetrazol-induced generalized seizures

The peptide angiotensin IV (Ang IV) influences seizure susceptibility in rat and mouse models. Indeed, Ang IV has been shown to protect rats from limbic seizures in the focal pilocarpine model. Moreover, both anticonvulsive and antiepileptogenic effects of Ang IV have been reported in the acute pentylenetetrazol (PTZ) and kindling model of generalized seizures in mice. It has been hypothesized that the latter effects on seizures could be established via a modulatory effect on dopamine receptors in the basal ganglia or via an indirect interaction between Ang IV and adenosine A1 receptors. However, a possible role for insulin-regulated aminopeptidase (IRAP), the high affinity binding site for Ang IV, has not been studied yet. To unequivocally unravel the involvement of IRAP in generalized seizure generation, we investigated the susceptibility of male IRAP wild-type (IRAP^+/+) and knock-out (IRAP^−/−) mice to PTZ-induced seizures. Challenging these mice intravenously with PTZ resulted in significantly increased thresholds for myoclonic twitch and generalized clonic seizures with loss of righting reflexes in IRAP^−/− mice compared to their IRAP^+/+ littermates. These behavioural data were confirmed by video-electrocorticography monitoring. Our study shows that IRAP^−/− mice are less sensitive to the development of PTZ-induced seizures and suggests that IRAP is involved in generalized seizure generation.     

New Mouse Model of Alzheimer’s

Amyloid β-peptide (Aβ) accumulation is a key characteristic of Alzheimer’s disease (AD); therefore, mouse models of AD exhibiting Aβ pathology are valuable tools for unraveling disease mechanisms. However, the overexpression of Aβ precursor protein (APP) used in previous mouse models may cause Aβ-independent artifacts that influence data interpretation. To circumvent these problems, we used an APP knock-in (KI) strategy to introduce mutations to the mouse APP gene to develop a new generation of AD mouse models. These new models, termed APP^NL-F and APP^NL-G-F, have endogenous APP levels and develop robust Aβ amyloidosis, which induce synaptic degeneration and memory impairments. Thus, we suggest that these novel APP KI mice will serve as important tools to elucidate molecular mechanisms of AD.     

In vitro modelling of Alzheimer's disease: degeneration and cell death induced by viral delivery of amyloid and tau

With increasing life expectancy, Alzheimer's disease (AD) and other dementias pose an increasing and as yet unresolved health problem. A variety of cellular models of AD has helped to decipher some key aspects of amyloid and tau related degeneration. The initial approach of extracellular applications of synthetic peptides has now been replaced by the introduction of amyloid precursor protein (APP) and tau genes. In the present study adenoviral transductions were exploited for gene delivery into primary rat hippocampal and dorsal root ganglion (DRG) cultures to enable comparative and mechanistic studies at the cellular level and subsequent drug testing. Time lapse experiments revealed a different pattern of cell death: apoptotic-like for APP whereas tau positive cells joined and formed clusters. Mutated human APP or tau expression caused accelerated neuronal damage and cell death (cf. EGFP: − 50% for APP at 5 days; − 40% for tau at 3 days). This reduction in viability was preceded by decreased excitability, monitored via responses to depolarising KCl-challenges in Ca²⁺ imaging experiments. Additionally, both transgenes reduced neurite outgrowth in DRG neurones. Treatment studies confirmed that APP induced-damage can be ameliorated by β- and γ-secretase inhibitors (providing protection to 60–100% of control levels), clioquinol (80%) and lithium (100%); while anti-aggregation treatments were beneficial for tau-induced damage (60–90% recovery towards controls). Interestingly, caffeine was the most promising drug candidate for therapeutic intervention with high efficacy in both APP (77%) and tau-induced models (72% recovery). Overall, these cellular models offer advantages for mechanistic studies and target identification in AD and related disorders.     

Intracellular acidification alters myogenic responsiveness and vasomotion of mouse middle cerebral arteries

Intracellular pH (pH_i) in the vascular wall modulates agonist-induced vasocontractile and vasorelaxant responses in mesenteric arteries, whereas effects on myogenic tone have been unsettled. We studied the role of Na⁺,HCO₃⁻ cotransporter NBCn1 in mouse isolated middle cerebral arteries and the influence of pH_i disturbances on myogenic tone. Na⁺,HCO₃⁻ cotransport was abolished in arteries from NBCn1 knockout mice and steady-state pH_i ∼0.3 units reduced compared with wild-type mice. Myogenic tone development was low under control conditions but increased on treatment with the NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME). This effect of L-NAME was smaller in arteries from NBCn1 knockout than wild-type mice. Myogenic tone with L-NAME present was significantly lower in arteries from NBCn1 knockout than wild-type mice and was abolished by rho-kinase inhibitor Y-27632. The arteries displayed vasomotion, and this rhythmic contractile pattern was also attenuated in arteries from NBCn1 knockout mice. No differences in membrane potential or intracellular [Ca²⁺] were seen between arteries from NBCn1 knockout and wild-type mice. We propose that NO production and rho-kinase-dependent Ca²⁺ sensitivity are reduced at low pH_i in pressurized mouse middle cerebral arteries. This likely impedes the ability to adjust to changes in perfusion pressure and regulate cerebral blood flow.     

Endogenous overproduction of β-amyloid induces tau hyperphosphorylation and decreases the solubility of tau in N2a cells

Summary. Although neurofibrillary tangles and senile plaques have been identified as the hallmark pathological changes in the brain of Alzheimer’s disease (AD), the relationship between them is still not fully understood. In the present study, we have studied the effect of endogenously overproduced amyloid β (Aβ) on tau by using wild type amyloid precursor protein (APP) transfected (N2a/APP695), or Swedish mutant APP plus Δ9 deleted presenilin-1 co-transfected (N2a/APPswe.Δ9) and APP vector transfected (N2a/vector) cell lines. We measured the secreted and intracellular Aβ, including Aβ_1–40 and Aβ_1–42, by Sandwich ELISA assay. It was shown that the levels of Aβ were increased time-dependently in N2a/APP695 and N2a/APPswe.Δ9 but not in N2a/vector upon butyric acid (BA) treatment. Compared with N2a/vector cells, tau in N2a/APP695 and N2a/APPswe.Δ9 cells was not extracted by RIPA buffer, and the SDS-extracted tau protein was hyperphosphorylated at Tau-1 and PHF-1 epitopes upon BA treatment. Obvious accumulation of the hyperphosphorylated tau in N2a/APP695 and N2a/APPswe.Δ9 cells was observed at 48 h after BA treatment. The total level of the extracted tau was reduced in N2a/APP695 and N2a/APPswe.Δ9 lines compared with N2a/vector cells by Western blot, and this reduction of total tau was also detected by immunofluorescence staining. No obvious alteration of tau mRNA was observed in both N2a/APP695 and N2a/APPswe.Δ9 cells compared with N2a/vector. This study provides direct evidence demonstrating that endogenously overproduced Aβ not only induces tau hyperphosphorylation but also decreases the level and solubility of tau in N2a cell lines. The first and second authors contributed equally to the paper