Effects of Cerebrolysin™ on neurogenesis in an APP transgenic model of Alzheimer’s disease

Cerebrolysin (CBL) is a peptide mixture with neurotrophic effects that might reduce the neurodegenerative alterations in Alzheimer’s disease (AD). We have previously shown that in the amyloid precursor protein (APP) transgenic (tg) mouse model of AD, CBL improves synaptic plasticity and behavioral performance. However, the mechanisms are not completely clear. The neuroprotective effects of CBL might be related to its ability to promote neurogenesis in the hippocampal subgranular zone (SGZ) of the dentate gyrus (DG). To study this possibility, tg mice expressing mutant APP under the Thy-1 promoter were injected with BrdU and treated with CBL for 1 and 3 months. Compared to non-tg controls, vehicle-treated APP tg mice showed decreased numbers of BrdU-positive (+) and doublecortin+ (DCX) neural progenitor cells (NPC) in the SGZ. In contrast, APP tg mice treated with CBL showed a significant increase in BrdU+ cells, DCX+ neuroblasts and a decrease in TUNEL+ and activated caspase-3 immunoreactive NPC. CBL did not change the number of proliferating cell nuclear antigen+ (PCNA) NPC or the ratio of BrdU+ cells converting to neurons and astroglia in the SGZ cells in the APP tg mice. Taken together, these studies suggest that CBL might rescue the alterations in neurogenesis in APP tg mice by protecting NPC and decreasing the rate of apoptosis. The improved neurogenesis in the hippocampus of CBL-treated APP tg mice might play an important role in enhancing synaptic formation and memory acquisition.     

Effects of triptolide on hippocampal microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer’s disease

The principal pathology of Alzheimer's disease includes neuronal extracellular deposition of amyloid-beta peptides and formation of senile pl aques,which in turn induce neuroinflammation in the brain.Triptolide,a natural extract from the vine-like herb Tripterygium wilfordii Hook F,has potent anti-inflammatory and immunosuppressive efficacy.Therefore,we determined if triptolide can inhibit activation and proliferation of microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer's disease.We used 1 or 5 μg/kg/d triptolide to treat APP/PS1 double transgenic mice(aged 4–4.5 months) for 45 days.Unbiased stereology analysis found that triptolide dose-dependently reduced the total number of microglial cells,and transformed microglial cells into the resting state.Further,triptolide(5 μg/kg/d) also reduced the total number of hippocampal astrocytes.Our in vivo test results indicate that triptolide suppresses activation and proliferation of microglial cells and astrocytes in the hippocampus of APP/PS1 double transgenic mice with Alzheimer's disease.     

Gender dependent APP processing in a transgenic mouse model of Alzheimer’s disease

Summary. Epidemiological studies have reported a higher prevalence and incidence of Alzheimer’s disease (AD) in women. The biochemical basis for this gender-disparate susceptibility is unknown. A gender effect on AD-typical plaque pathology has been shown in APP transgenic mouse models of AD. Female mice elicit higher plaque load than male mice. In an effort to analyze gender-dependent APP processing during postnatal development, we examined APP transgenic mice at time points prior to plaque deposition. At 14 weeks of age there was a significant elevation of C99 and Aβ in female mice compared to males. Furthermore we observed a slight decrease of BACE-activity in male mice as well as higher cerebral manganese levels in females. Although the decline in estrogen levels due to menopause in female patients is still discussed to be a risk factor for AD our results implicates that additional factors like modified BACE-activity or metal levels may contribute to the higher prevalence and incidence of AD in females.     

IL-6 trans-signaling in the brain influences the behavioral and physio-pathological phenotype of the Tg2576 and 3xTgAD mouse models of Alzheimer’s disease

Alzheimer’s disease (AD) is the most commonly diagnosed dementia but its underlying pathological mechanisms still unclear. Neuroinflammation and secretion of cytokines such as interleukin-6 (IL-6) accompany the main hallmarks of the disease: amyloid plaques and neurofibrillary tangles. In this study, we analyzed the role of IL-6 trans-signaling in two mouse models of AD, Tg2576 and 3xTg-AD mice. The inhibition of IL-6 trans-signaling partially rescued the AD-induced mortality in females of both models. Before amyloid plaques deposition, it reversed AD-induced changes in exploration and anxiety (but did not affect locomotion) in Tg2576 female mice. However, after plaque deposition the only behavioral trait affected by the inhibition of IL-6 trans-signaling was locomotion. Results in the Morris water maze suggest that cognitive flexibility was reduced by the blocking of the IL-6 trans-signaling in young and old Tg2576 female mice. The inhibition of IL-6 trans-signaling also decreased amyloid plaque burden in cortex and hippocampus, and Aβ₄₀ and Aβ₄₂ levels in the cortex, of Tg2576 female mice. The aforementioned changes might be correlated with changes in blood vessels and matrix structure and organization rather than changes in neuroinflammation. 3xTgAD mice showed a very mild phenotype regarding amyloid cascade, but results were in accordance with those of Tg2576 mice. These results strongly suggest that the inhibition of the IL-6 trans-signaling could represent a powerful therapeutic target in AD.     

M1 muscarinic agonists can modulate some of the hallmarks in Alzheimer’s disease

M₁ muscarinic receptors (M₁ mAChRs) play a role in an apparent linkage of three major hallmarks of Alzheimer’s disease (AD): β-amyloid (Aβ) peptide; tau hyperphosphorylation and paired helical filaments (PHFs); and loss of cholinergic function conducive to cognitive impairments. We evaluated the M₁ muscarinic agonists AF102B (Cevimeline, EVOXAC?: prescribed for Sjøgren’s syndrome), AF150(S), and AF267B on some of these hallmarks of AD. Activation of M₁ mAChRs with these agonists leads, inter alia, to enhanced secretion of amyloid precursor protein (α-APP), (via α-secretase activation), to decreased Aβ (via γ-secretase inhibition), and to inhibition of Aβ- and/or oxidative stress-induced cell death. In several animal models mimicking different aspects of AD, these drugs restored cognitive impairments, and in select cases induced a decrease in brain Aβ elevation, with a high safety margin, following po administration. Notably, in mice with small hippocampi, unlike rivastigmine and nicotine, AF150(S) and AF267B restored cognitive impairments also on escape latency in a Morris water maze paradigm, in reversal learning. Studies from other labs showed that AF102B and talsaclidine (another M₁ agonist) decreased cerbrospinal fluid (CSF) Aβ in AD patients following chronic treatment, being the first reported drugs with such a profile. The clinical significance of these studies remains to be elucidated, yet based on in vivo (rabbits) and in vitro studies (cell cultures), our M₁ agonists can decrease brain Aβ, owing to a novel and dual complementary effect (e.g., inhibition of γ-secretase and activation of α-secretase). Remarkably, although M₁ agonists can decrease CSF Aβ in AD patients, an increased AD-type pathology in Parkinson’s disease was recently been associated with chronic antimuscarinic treatment. In another aspect, these agonists decreased tau hyperphosphorylation in vitro and in vivo. Notably, nicotinic agonists or cholinesterase inhibitors increased tau hyperphosphorylation. In summary, the M₁ agonists tested are effective on cognition and behavior and show unique disease-modifying properties owing to beneficial effects on major hallmarks of AD. This may place such drugs in the first line of modern AD therapies (e.g., β- or γ-secretase inhibitors, vaccines against Aβ, statins, and inhibitors of tau hyperphosphorylation).     

Clustering of plaques contributes to plaque growth in a mouse model of Alzheimer’s disease

Amyloid-β (Aβ) plaque deposition plays a central role in the pathogenesis of Alzheimer’s disease (AD). Post-mortem analysis of plaque development in mouse models of AD revealed that plaques are initially small, but then increase in size and become more numerous with age. There is evidence that plaques can grow uniformly over time; however, a complementary hypothesis of plaque development is that small plaques cluster and grow together thereby forming larger plaques. To investigate the latter hypothesis, we studied plaque formation in APPPS1 mice using in vivo two-photon microscopy and immunohistochemical analysis. We used sequential pre- and post-mortem staining techniques to label plaques at different stages of development and to detect newly emerged plaques. Post-mortem analysis revealed that a subset (22 %) of newly formed plaques appeared very close (<40 μm) to pre-existing plaques and that many close plaques (25 %) that were initially separate merged over time to form one single large plaque. Our results suggest that small plaques can cluster together, thus forming larger plaques as a complementary mechanism to simple uniform plaque growth from a single initial plaque. This study deepens our understanding of Aβ deposition and demonstrates that there are multiple mechanisms at play in plaque development.     

Axonopathy in an APP/PS1 transgenic mouse model of Alzheimer’s disease

While axonopathy is a prominent feature in a variety of neurodegenerative diseases, it has been largely neglected in Alzheimer’s disease (AD), despite the observation of frequent motoric deficits in AD patients. In the present report we used transgenic mice overexpressing human mutant β-amyoid precursor protein (APP^751SL) and presenilin-1 (PS1^M146L) that exhibit elevated intraneuronal Aβ42 levels. We observed abundant age-dependent axonopathy in the spinal cord: axons immunopositive for ubiquitin in the dorsal column; axonal swellings (spheroids) which accumulated APP, neurofilament, and ubiquitin; as well as myelin ovoid structures, which serve as markers for nerve fiber degeneration in both white and gray matter. Both descending and ascending axonal tracts in white matter were affected. Neuritic plaques also developed in an age-dependent manner starting in the cervical region. Furthermore, early intraneuronal Aβ was detected in some but not all motor neurons before plaque formation. In the present APP/PS1 transgenic mouse model we could show for the first time that elevated intracellular Aβ levels lead to an axonopathy characterized by the formation of axonal spheroids and myelin ovoids. The same pathological alterations are known from AD patients or transgenic models overexpressing Tau or ApoE, however, these disturbances in axonal transport occur in the absence of any signs of concomitant Tau pathology. This strengthens the prevailing amyloid hypothesis as a primary trigger of AD-typical pathological alterations.Oliver Wirths and Joachim Weis contributed equally.     

Aquaporin 4 deficiency eliminates the beneficial effects of voluntary exercise in a mouse model of Alzheimer’s disease

Regular exercise has been shown to reduce the risk of Alzheimer’s disease(AD).Our previous study showed that the protein aquaporin 4(AQP4),which is specifically expressed on the paravascular processes of astrocytes,is necessary for glymphatic clearance of extracellular amyloid beta(Aβ)from the brain,which can delay the progression of Alzheimer’s disease.However,it is not known whether AQP4-regulated glymphatic clearance of extracellular Aβis involved in beneficial effects of exercise in AD patients.Our results showed that after 2 months of voluntary wheel exercise,APP/PS1 mice that were 3 months old at the start of the intervention exhibited a decrease in Aβburden,glial activation,perivascular AQP4 mislocalization,impaired glymphatic transport,synapse protein loss,and learning and memory defects compared with mice not subjected to the exercise intervention.In contrast,APP/PS1 mice that were 7 months old at the start of the intervention exhibited impaired AQP4 polarity and reduced glymphatic clearance of extracellular Aβ,and the above-mentioned impairments were not alleviated after the 2-month exercise intervention.Compared with age-matched APP/PS1 mice,AQP4 knockout APP/PS1 mice had more serious defects in glymphatic function,Aβplaque deposition,and cognitive impairment,which could not be alleviated after the exercise intervention.These findings suggest that AQP4-dependent glymphatic transport is the neurobiological basis for the beneficial effects of voluntary exercises that protect against the onset of AD.     

Rethinking on the concept of biomarkers in preclinical Alzheimer’s disease

The neuropathological processes eventually leading to Alzheimer’s disease (AD) are thought to start decades before the appearance of clinical symptoms and the clinical diagnosis of AD dementia. The term “preclinical AD” has been recently introduced to identify this “silent stage” of AD, when the disease is already present, but symptoms are not yet clinically evident. Advances in AD biomarkers have dramatically improved the ability to detect AD pathological processes in vivo in cognitively intact subjects, thus demonstrating the presence of AD pathology in the preclinical phase. This review focuses on the recent advances in the field of neuroimaging and CSF AD biomarkers specifically in the preclinical phase of AD, and aims to discuss the significance that such biomarkers could have in cognitively intact subjects. Even though the use of such biomarkers in AD preclinical phase has contributed to improve our understanding of AD early pathological processes, it raised also a number of new challenges that still remain to be overcome, such as a better definition of the clinical and individual significance of currently known biomarkers in preclinical stages and the development of novel biomarkers of different early AD-related events.     

DNA fragmentation, gliosis and histological hallmarks of Alzheimer’s disease

The extent of DNA fragmentation analysed using the TUNEL technique was evaluated in post-mortem human brain tissue. Twenty-four patients with clinical and histopathological diagnosis of Alzheimer’s disease (AD) and a short post-mortem delay were analysed. We report an increase in the count of TUNEL-labelled cells as the pathology of AD intensifies. Our results point out a significant correlation between neurofibrillary tangle and senile/neuritic plaque score and TUNEL-labelled cells. Patients with two copies of apolipoprotein (Apo) E ɛ4 allele had highest number of histopathological hallmarks lesions of AD, whereas the ApoE genotype did not significantly influence the density of TUNEL-positive cells. No significant correlation was found between β-amyloid protein load and TUNEL-labelled cells. There was no relationship between the age at death, age at onset, extent of astrogliosis or microgliosis and TUNEL-labelled cells in our material. Received: 23 November 1999 / Revised: 18 January 2000, 22 February2000 / Accepted: 22 February 2000     

Genome-wide analysis of DNA methylation in an APP/PS1 mouse model of Alzheimer’s disease

To investigate aberrant genome-wide CpG methylation patterns in cortex brain tissue of APP/PS1 mice and as compared to controls, which allows for identification of novel disease-associated genes. This study investigates the genome-wide DNA methylation profiles of the cortex from APP/PS1 transgenic mice and control mice using the Roche NimbleGen chip platform. Functional analysis was then conducted by Ingenuity Pathways Analysis system. The methylated DNA fragments in the genome of each sample were enriched by MeDIP and the whole-genome interrogations were hybridized to the Roche NimbleGen Human DNA Methylation 3x720 K CpG Island Plus RefSeq Promoter Array that cover 15,980 CpG islands and 20,404 reference gene promoter regions of the entire human genome. Analysis reveals 2346 CpG sites representing 485 unique genes as potentially associated with AD disease status pending confirmation in additional study. At the same time, these hyper-methylated genes display familial aggregation. An impairment of the transforming growth factor-β1 (TGF-β1) signaling pathway has been demonstrated to be specific to the AD brain and, particularly, to the early phase of the disease, supporting a role for epigenetic change of TGF-β1 in AD pathology. In future research, we will focus on TGF-β1, as it appeared to be the most promising candidate for AD.     

Apolipoprotein E dose-dependent modulation of β-amyloid deposition in a transgenic mouse model of Alzheimer’s disease

Susceptibility to the development of Alzheimer’s disease (AD) is increased for individuals harboring one or more apolipoprotein E4 (apoE4) alleles. Even though several isoform-specific effects of apoE have been identified, the relationship between biochemical function and risk factor assessment remains unknown. Our previous studies have demonstrated that there is an equilibrium between cerebral spinal fluid (CSF) and plasma β-amyloid (Aβ) and that amyloid plaques can modify this equilibrium. Trafficking of soluble central nervous system (CNS) Aβ is a very dynamic system that almost certainly is modulated by Aβ-binding proteins. Altered trafficking of the Aβ peptide might have a dramatic consequence as to whether the peptide is metabolized or begins to deposit within the brain. To gain a better understanding of the molecular mechanisms by which apoE influences AD pathogenesis and/or Aβ trafficking, we developed PDAPP transgenic mice that express different levels of human apoE3. Analysis of the soluble CNS pools of Aβ in young mice showed an apoE3 dose-dependent decrease in Aβ levels (E3^−/−>E3^−/−>E3^+/+). In addition to the dose-dependent effects on soluble Aβ, by 15 mo of age there were highly significant differences in the amount of deposited Aβ between the genotypes (E3^−/−>E3^−/−>E3^+/+). These data indicate that apoE3 provides a dose-dependent protective effect against Aβ deposition. This study suggests that increasing human apoE levels in brain might be a possible therapeutic target for preventing AD.     

Variations in the neuropathology of familial Alzheimer’s disease

Mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes cause autosomal dominant familial Alzheimer’s disease (AD). PSEN1 and PSEN2 are essential components of the γ-secretase complex, which cleaves APP to affect Aβ processing. Disruptions in Aβ processing have been hypothesised to be the major cause of AD (the amyloid cascade hypothesis). These genetic cases exhibit all the classic hallmark pathologies of AD including neuritic plaques, neurofibrillary tangles (NFT), tissue atrophy, neuronal loss and inflammation, often in significantly enhanced quantities. In particular, these cases have average greater hippocampal atrophy and NFT, more significant cortical Aβ42 plaque deposition and more substantial inflammation. Enhanced cerebral Aβ40 angiopathy is a feature of many cases, but particularly those with APP mutations where it can be the dominant pathology. Additional frontotemporal neuronal loss in association with increased tau pathology appears unique to PSEN mutations, with mutations in exons 8 and 9 having enlarged cotton wool plaques throughout their cortex. The mechanisms driving these pathological differences in AD are discussed.     

Aberrant stress response associated with severe hypoglycemia in a transgenic mouse model of Alzheimer’s disease

Patients with Alzheimer’s disease (AD) exhibit alterations in glucose metabolism and dysregulation of the stress-responsive hypothalamic-pituitary-adrenal (HPA) neuroendocrine system. The mechanisms responsible for these alterations and their possible contributions to the neurodegenerative process in AD are unknown. We now report that transgenic mice expressing a mutant form of human amyloid precursor protein (APP) that causes inherited early-onset AD exhibit increased sensitivity to physiological stressors, which is associated with aberrancies in HPA function and regulation of blood glucose levels. Specifically, APP mutant mice exhibit severe hypoglycemia and death following food restriction, and sustained elevations of plasma glucocorticoid levels and hypoglycemia following restraint stress. The alterations in HPA function and glucose regulation were evident in relatively young mice prior to overt deposition of amyloid β-peptide (Aβ). However, diffuse accumulations of Aβ were present in the hypothalamus of older mice, suggesting a role for soluble forms of Aβ in dysregulation of HPA function. Our data demonstrate disturbances in neuroendocrine function in APP mutant mice similar to those seen in AD patients. These impairments in stress response, glucocorticoid signaling, and regulation of blood glucose should be considered in interpretations of data from past and future studies of APP mutant mice. These authors contributed equally to this work     

MiR-206 decreases brain-derived neurotrophic factor levels in a transgenic mouse model of Alzheimer’s disease

MicroRNA alterations have been reported in patients with Alzheimer’s disease (AD) and AD mouse models. We now report that miR-206 is upregulated in the hippocampal tissue, cerebrospinal fluid, and plasma of embryonic APP/PS1 transgenic mice. The increased miR-206 downregulates the expression of brain-derived neurotrophic factor (BDNF). BDNF is neuroprotective against cell death after various insults, but in embryonic and newborn APP/PS1 mice it is decreased. Thus, a specific microRNA alteration may contribute to AD pathology by downregulating BDNF.     

Daicong solution effects on brain ultrastructure in a rat model of Alzheimer’s disease

BACKGROUND： Infusion of kainic acid into the basal nuclei induces neuronal excitotoxicity, degeneration and necrosis, resulting in disturbed learning and memory functions. OBJECTIVE： To explore the effects of different doses of traditional Chinese medicine Daicong solution on brain ultrastructure in a rat model of Alzheimer＇s disease. DESIGN, TIME AND SETTING： The randomized, controlled, cellular morphology experiment was performed at the Shandong Provincial Key Laboratory of Molecular Immunology of Weifang Medical University, China from October 2006 to March 2007. MATERIALS： Fifty healthy, Sprague Dawley rats, aged 22-months, were used to establish rat models of Alzheimer＇s disease. The Morris water maze was prepared at the Pharmacometrics Key Laboratory of Weifang Medical University in Shandong Province of China. Traditional Chinese medicine Daicong solution （crude drug 1 g/mL）, composed of radix ginseng, rehmannia dried rhizome, anemarrhenae and radix astragali, was produced by the Department of Pharmacy of Hospital Affiliated to Weifang Medical University. Kainic acid was provided by Professor Xiuyan Li from Weifang Medical University. METHODS： A total of 40 model rats were equally and randomly divided into four groups： dementia model, low-dose Daicong solution （5 g/kg/d）, moderate-dose Daicong solution （10 g/kg/d）, and high-dose Daicong solution （20 g/kg/d）. An additional 10 healthy rats served as the normal control group. Rats in the dementia model and normal control groups received saline （10 mL/kg/d）. MAIN OUTCOME MEASURES： Neural cell ultrastructure was observed utilizing electron microscopy after 1 month of respective treatments. RESULTS： Compared with the normal control group, electron density and the number of ribosomes were significantly reduced in neuronal cytoplasm, and many lipofuscin grains and vacuole-like changes were observed in mitochondria in the dementia model group. In addition, nuclear chromatin presented with different sizes of plaque-shaped degene     

Omega-3 polyunsaturated fatty acids promote brain-to-blood clearance of β-Amyloid in a mouse model with Alzheimer’s disease

Amyloid-β (Aβ) plaques is one of the typical pathological hallmark of Alzheimer disease (AD). Accumulating evidence suggests that the imbalance between Aβ production and clearance leads to extracellular Aβ accumulation in the brain. It is reported that the blood–brain barrier (BBB) transport plays a predominant role in Aβ clearance from brain to blood. In the present study, we investigated dynamic alterations of BBB transport function in the early disease stage of AD using APPswe/PS1dE9 C57BL/6J (APP/PS1) transgenic mice. Our results showed that the expression of lipoprotein receptor-related protein 1 (LRP-1), a main efflux transporter of BBB, started to decrease at the age of 4 months old. Interestingly, supplementing with fish oil which is rich in omega-3 polyunsaturated fatty acids (PUFAs) significantly enhanced the expression level of LRP-1 and promoted Aβ clearance from the bran to circulation, as revealed by reduced soluble/insoluble Aβ levels and senile plaques in the brain parenchyma and a corresponding increase of Aβ levels in plasma. Besides, fish oil supplement significantly inhibited the NF-κB activation, reduced the expression of interleukin-1β and tumor necrosis factor-α, and suppressed the glial activation in APP/PS1 mice. The results of the study provide evidence that BBB transport function could be impaired at a very early disease stage, which might contribute to Aβ pathological accumulation in AD, and omega-3 PUFAs intervention could be an effective strategy for the prevention of the progression of AD through promoting Aβ clearance from brain-to-blood.     

Neuroinflammatory perspectives on the two faces of Alzheimer’s disease

Summary. The amyloid plaques in Alzheimers disease (AD) brains are co-localized with a broad variety of inflammation-related proteins (complement factors, acute-phase proteins, pro-inflammatory cytokines) and clusters of activated microglia. The present data suggest that A deposits in AD brains are closely associated with a locally induced, non-immune mediated, chronic inflammatory response. Clinicopathological and neuroradiological studies show that activation of microglia is a relatively early pathogenic event that precedes the process of neuropil destruction in AD. Epidemiological studies indicate that polymorphisms of certain cytokines and acute-phase proteins that are colocalized with A plaques, are genetic risk factors of AD. Epidemiological studies have also shown that the use of classical nonsteroidal anti-inflammatory drugs (NSAIDs) can prevent the risk of AD but clinical trials with anti-inflammatory drugs in AD patients were negative. These findings indicate that anti-inflammatory agents can be helpful in the prevention but not in the treatment of AD. So, pathological, genetic and therapeutic studies suggest that inflammatory mechanisms are most likely involved in the early steps of the pathological cascade. In the autosomal dominant inherited forms of AD the primary factor is the increased production of A1–42 resulting into fibrillar A deposition that elicits a brain inflammatory response. The etiology of the sporadic forms is yet unknown but this subtype is considered to be heterogeneous and multifactorial in its pathogenesis. Here we review the evidence that inflammation related events could be a critical etiological factor in certain forms of the sporadic AD.