Apolipoprotein E deposition and astrogliosis are associated with maturation of β-amyloid plaques in βAPPswe transgenic mouse: Implications for the pathogenesis of Alzheimer’s disease

A transgenic mouse expressing the human β-amyloid precursor protein with the ‘Swedish’ mutation, Tg2576, was used to investigate the mechanism of β-amyloid (Aβ) deposition. Previously, we have reported that the major species of Aβ in the amyloid plaques of Tg2576 mice are Aβ_1-40 and Aβ_1-42. Moreover, Aβ_1-42 deposition precedes Aβ_1-40 deposition, while Aβ_1-40 accumulates in the central part of the plaques later in the pathogenic process. Those data indicate that Aβ deposits in Tg2576 mice have similar characteristics to those in Alzheimer’s disease. In the present study, to understand more fully the amyloid deposition mechanism implicating Alzheimer’s disease pathogenesis, we examined immunohistochemically the distributions of apolipoprotein E (apoE) and Aβ in amyloid plaques of aged Tg2576 mouse brains. Our findings suggest that Aβ_1-42 deposition precedes apoE deposition, and that Aβ_1-40 deposition follows apoE deposition during plaque maturation. We next examined the relationship between apoE and astrogliosis associated with amyloid plaques using a double-immunofluorescence method. Extracellular apoE deposits were always associated with reactive astrocytes whose processes showed enhancement of apoE-immunoreactivity. Taken together, the characteristics of amyloid plaques in Tg2576 mice are similar to those in Alzheimer’s disease with respect to apoE and astrogliosis. Furthermore, apoE deposition and astrogliosis may be necessary for amyloid plaque maturation.     

Hippocampal estrogen β-receptor immunoreactivity is increased in Alzheimer’s disease

Post-menopausal estrogen use reduces the risk and severity of Alzheimer’s disease (AD). The present study investigates the distribution of both estrogen receptors ERα and ERβ in the human hippocampus in aged controls and in AD cases with immunohistochemistry. No ERα immunoreactivity was observed both in controls and in AD cases. On the other hand, ERβ was observed in some neuronal cells in the hippocampal subfields CA1–4, in astrocytes and in extracellular deposits both in controls and AD cases. The ERβ immunoreactivity was distinctly increased in all AD cases in cellular and extracellular localizations indicating a role for ERβ-mediated estrogen effects in AD-related neuropathology. This study provides the first demonstration of ERβ in human hippocampus in aged controls compared to AD cases.     

Genetic reductions of β‐site amyloid precursor protein‐cleaving enzyme 1 and amyloid‐β ameliorate impairment of conditioned taste aversion memory in 5XFAD Alzheimer’s disease model mice

Although transgenic mouse models of Alzheimer’s disease (AD) recapitulate amyloid‐β (Aβ)‐related pathologies and cognitive impairments, previous studies have mainly evaluated their hippocampus‐dependent memory dysfunctions using behavioral tasks such as the water maze and fear conditioning. However, multiple memory systems become impaired in AD as the disease progresses and it is important to test whether other forms of memory are affected in AD models. This study was designed to use conditioned taste aversion (CTA) and contextual fear conditioning paradigms to compare the phenotypes of hippocampus‐independent and ‐dependent memory functions, respectively, in 5XFAD amyloid precursor protein/presenilin‐1 transgenic mice that harbor five familial AD mutations. Although both types of memory were significantly impaired in 5XFAD mice, the onset of CTA memory deficits (～9 months of age) was delayed compared with that of contextual memory deficits (～6 months of age). Furthermore, 5XFAD mice that were genetically engineered to have reduced levels of β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) (BACE1^+/?·5XFAD) exhibited improved CTA memory, which was equivalent to the performance of wild‐type controls. Importantly, elevated levels of cerebral β‐secretase‐cleaved C‐terminal fragment (C99) and Aβ peptides in 5XFAD mice were significantly reduced in BACE1^+/?·5XFAD mice. Furthermore, Aβ deposition in the insular cortex and basolateral amygdala, two brain regions that are critically involved in CTA performance, was also reduced in BACE1^+/?·5XFAD compared with 5XFAD mice. Our findings indicate that the CTA paradigm is useful for evaluating a hippocampus‐independent form of memory defect in AD model mice, which is sensitive to rescue by partial reductions of the β‐secretase BACE1 and consequently of cerebral Aβ.     

Small molecule β‐amyloid inhibitors that stabilize protofibrillar structures in vitro improve cognition and pathology in a mouse model of Alzheimer’s disease

β‐Amyloid (Aβ) peptides are thought to play a major role in the pathogenesis of Alzheimer’s disease. Compounds that disrupt the kinetic pathways of Aβ aggregation may be useful in elucidating the role of oligomeric, protofibrillar and fibrillar Aβ in the etiology of the disease. We have previously reported that scyllo‐inositol inhibits Aβ₄₂ fibril formation but the mechanism(s) by which this occurs has not been investigated in detail. Using a series of scyllo‐inositol derivatives in which one or two hydroxyl groups were replaced with hydrogen, chlorine or methoxy substituents, we examined the role of hydrogen bonding and hydrophobicity in the structure–function relationship of scyllo‐inositol–Aβ binding. We report here that all scyllo‐inositol derivatives demonstrated reduced effectiveness in preventing Aβ₄₂ fibrillization compared with scyllo‐inositol, suggesting that scyllo‐inositol interacts with Aβ₄₂ via key hydrogen bonds that are formed by all hydroxyl groups. Increasing the hydrophobicity of scyllo‐inositol by the addition of two methoxy groups (1,4‐di‐O‐methyl‐scyllo‐inositol) produced a derivative that stabilized Aβ₄₂ protofibrils in vitro. Prophylactic administration of 1,4‐di‐O‐methyl‐scyllo‐inositol to TgCRND8 mice attenuated spatial memory impairments and significantly decreased cerebral amyloid pathology. These results suggest that Aβ aggregation can be targeted at multiple points along the kinetic pathway for the improvement of Alzheimer’s disease‐like pathology.     

CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the α6/β1 integrin and the neural adhesion molecule L1

We describe here a novel monoclonal antibody (mab INTRODUCTION H6) which recognizes CD9, an integral cell surface constituent previously described in cells of the hematopoietic lineage and involved in the aggregation of platelets. Mab H6 was raised against membranes of immature mouse astrocytes and reacted with a protein of 25–27 kD in detergent extracts of adult mouse brain membranes. Sequence analysis of the N-terminal amino acids revealed an identity of 96% with CD9 from mouse kidney. CD9 was localized in the central and peripheral mouse nervous systems: in the spinal cord of 11-day-old mouse embryos, CD9 was strongly expressed in the floor and roof plates. In the adult mouse sciatic nerve, myelin sheaths were highly CD9immunoreactive. Mab H6 reacted with the cell surfaces of both glial cells and neurons in culture and inhibited migration of neuronal cell bodies, neurite fasciculation and outgrowth of astrocytic processes from cerebellar microexplants. Neurite outgrowth from isolated small cerebellar neurons was increased in the presence of mab H6 on substrate-coated laminin, but not on substrate-coated poly-L-lysine. Addition of mab H6 elicited an increase in intracellular Ca²⁺ concentration in these cells on substrate-coated laminin. Immunoprecipitates of CD9 from cultured mouse neuroblastoma N2A cells contained the α6/β1 integrin. Moreover, preparations of CD9 immunoaffinity-purified from adult mouse brain using a mab H6 column contained the neural adhesion molecule L1, but not other neural adhesion molecules. CD9 bound to L1, but not to NCAM or MAG. Both the α6/β1 integrin and L1 could be induced to coredistribute with CD9 on the surface of cultured neuroblastoma N2A cells. The combined observations suggest that CD9 can associate with L1 and α6/β1 integrin to influence neural cell interactions in vitro. © 1996 Wiley-Liss, Inc.     

Epistasis between tau phosphorylation regulating genes (CDK5R1 and GSK‐3β) and Alzheimer’s disease risk

Objective – Glycogen synthase kinase‐3β (GSK‐3β) and cyclin‐dependent kinase 5 (CDK5) have been implicated as two major protein kinases involved in the abnormal hyperphosphorylation of tau in Alzheimer’s disease (AD) brain, and the development of neurofibrillary tangles. CDK5 regulatory subunit 1 (CDK5R1) encodes for p35, a protein required for activation of CDK5. As both CDK5R1 and GSK‐3β genes are related to phosphorylation of tau, we examined the combined contribution of these genes to the susceptibility for AD. Methods – In a case–control study in 283 AD patients and 263 healthy controls, we examined the combined effects between CDK5R1 (3′‐UTR, rs735555) and GSK‐3β (?50, rs334558) polymorphisms on susceptibility to AD. Results – Subjects carrying both the CDK5R1 (3′‐UTR, rs735555) AA genotype and the GSK‐3β (?50, rs334558) CC genotype had a 12.5‐fold decrease in AD risk (adjusted by age, sex and APOE status OR = 0.08, 95% CI = 0.01–0.76, P = 0.03), suggesting synergistic effects (epistasis) between both genes. Conclusion – These data support a role for tau phosphorylation regulating genes in risk for AD.     

Review: Sporadic Parkinson's disease: development and distribution of α‐synuclein pathology

The development of α‐synuclein immunoreactive aggregates in selectively vulnerable neuronal types of the human central, peripheral, and enteric nervous systems is crucial for the pathogenesis of sporadic Parkinson's disease. The presence of these lesions persists into the end phase of the disease, a process that is not subject to remission. The initial induction of α‐synuclein misfolding and subsequent aggregation probably occurs in the olfactory bulb and/or the enteric nervous system. Each of these sites is exposed to potentially hostile environmental factors. Once formed, the aggregates appear to be capable of propagating trans‐synaptically from nerve cell to nerve cell in a virtually self‐promoting pathological process. A regional distribution pattern of aggregated α‐synuclein emerges that entails the involvement of only a few types of susceptible and axonally interconnected projection neurons within the human nervous system. One major route of disease progression may originate in the enteric nervous system and retrogradely reach the dorsal motor nucleus of the vagal nerve in the lower brainstem. From there, the disease process proceeds chiefly in a caudo‐rostral direction through visceromotor and somatomotor brainstem centres to the midbrain, forebrain, and cerebral cortex. Spinal cord centres may become involved by means of descending projections from involved lower brainstem nuclei as well as by sympathetic projections connecting the enteric nervous system with postganglionic peripheral ganglia and preganglionic nuclei of the spinal cord. The development of experimental cellular and animal models is helping to explain the mechanisms of how abnormal α‐synuclein can undergo aggregation and how transmission along axonal connectivities can occur, thereby encouraging the initiation of potential disease‐modifying therapeutic strategies for sporadic Parkinson's disease.     

α-Synuclein-positive structures in cases with sporadic Alzheimer’s disease: morphology and its relationship to tau aggregation

Alzheimer’s disease (AD) and Parkinson’s disease share common clinical and pathological features. In this study, we examined the relationship between AD pathology and α-synuclein aggregation. The frequency and distribution of α-synuclein-positive structures were systematically investigated in 27 cases with sporadic AD by α-synuclein immuno-histochemistry. Thirteen (48.2%) of 27 cases had various α-synuclein-positive structures as well as Lewy bodies. The frequency and density of senile plaques and neurofibrillary tangles were not significantly different between cases with α-synuclein structures and those without. α-Synuclein-positive structures were found most frequently in the amygdala. The α-synuclein-positive inclusions that are different from Lewy bodies were observed at the highest rate in the hippocampus. The discovery of α-synuclein as the constituent of Lewy bodies facilitated the detection of Lewy-related structures even in AD cases with widespread and numerous neurofibrillary tangles. α-Synuclein-positive inclusions except for Lewy bodies are exposed, and the distribution of them indicates that Lewy body formation may be influenced by the degree of tau aggregation. This study also supports the suggestion that cases with AD pathology can be classified into two groups according to the existence or absence of α-synuclein aggregation.     

Phosphorylated α‐synuclein in the retina is a biomarker of Parkinson's disease pathology severity

Background : PD patients often have visual alterations, for example, loss of visual acuity, contrast sensitivity or motion perception, and diminished electroretinogram responses. PD pathology is mainly characterized by the accumulation of pathological α‐synuclein deposits in the brain, but little is known about how synucleinopathy affects the retina. Objective : To study the correlation between α‐synuclein deposits in the retina and brain of autopsied subjects with PD and incidental Lewy body disease. Methods : We evaluated the presence of phosphorylated α‐synuclein in the retina of autopsied subjects with PD (9 subjects), incidental Lewy body disease (4 subjects), and controls (6 subjects) by immunohistochemistry and compared the retinal synucleinopathy with brain disease severity indicators. Results : Whereas controls did not show any phosphorylated α‐synuclein immunoreactivity in their retina, all PD subjects and 3 of 4 incidental Lewy body disease subjects had phosphorylated α‐synuclein deposits in ganglion cell perikarya, dendrites, and axons, some of them resembling brain Lewy bodies and Lewy neurites. The Lewy‐type synucleinopathy density in the retina significantly correlated with Lewy‐type synucleinopathy density in the brain, with the Unified Parkinson's disease pathology stage and with the motor UPDRS. Conclusion : These data suggest that phosphorylated α‐synuclein accumulates in the retina in parallel with that in the brain, including in early stages preceding development of clinical signs of parkinsonism or dementia. Therefore, the retina may provide an in vivo indicator of brain pathology severity, and its detection could help in the diagnosis and monitoring of disease progression. © 2018 International Parkinson and Movement Disorder Society     

Memantine lowers amyloid‐β peptide levels in neuronal cultures and in APP/PS1 transgenic mice

Memantine is a moderate‐affinity, uncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist that stabilizes cognitive, functional, and behavioral decline in patients with moderate to severe Alzheimer's disease (AD). In AD, the extracellular deposition of fibrillogenic amyloid‐β peptides (Aβ) occurs as a result of aberrant processing of the full‐length Aβ precursor protein (APP). Memantine protects neurons from the neurotoxic effects of Aβ and improves cognition in transgenic mice with high brain levels of Aβ. However, it is unknown how memantine protects cells against neurodegeneration and affects APP processing and Aβ production. We report the effects of memantine in three different systems. In human neuroblastoma cells, memantine, at therapeutically relevant concentrations (1–4 μM), decreased levels of secreted APP and Aβ_1–40. Levels of the potentially amylodogenic Aβ_1–42 were undetectable in these cells. In primary rat cortical neuronal cultures, memantine treatment lowered Aβ_1–42 secretion. At the concentrations used, memantine treatment was not toxic to neuroblastoma or primary cultures and increased cell viability and/or metabolic activity under certain conditions. In APP/presenilin‐1 (PS1) transgenic mice exhibiting high brain levels of Aβ_1–42, oral dosing of memantine (20 mg/kg/day for 8 days) produced a plasma drug concentration of 0.96 μM and significantly reduced the cortical levels of soluble Aβ_1–42. The ratio of Aβ_1–40/Aβ_1–42 increased in treated mice, suggesting effects on the γ‐secretase complex. Thus, memantine reduces the levels of Aβ peptides at therapeutic concentrations and may inhibit the accumulation of fibrillogenic Aβ in mammalian brains. Memantine's ability to preserve neuronal cells against neurodegeneration, to increase metabolic activity, and to lower Aβ level has therapeutic implications for neurodegenerative disorders. © 2009 Wiley‐Liss, Inc.     

Mid‐life environmental enrichment increases synaptic density in CA1 in a mouse model of Aβ‐associated pathology and positively influences synaptic and cognitive health in healthy ageing

Early‐life cognitive enrichment may reduce the risk of experiencing cognitive deterioration and dementia in later‐life. However, an intervention to prevent or delay dementia is likely to be taken up in mid to later‐life. Hence, we investigated the effects of environmental enrichment in wildtype mice and in a mouse model of Aβ neuropathology (APP_SWE/PS1_dE9) from 6 months of age. After 6 months of housing in standard laboratory cages, APP_SWE/PS1_dE9 (n = 27) and healthy wildtype (n = 21) mice were randomly assigned to either enriched or standard housing. At 12 months of age, wildtype mice showed altered synaptic protein levels and relatively superior cognitive performance afforded by environmental enrichment. Environmental enrichment was not associated with alterations to Aβ plaque pathology in the neocortex or hippocampus of APP_SWE/PS1_dE9 mice. However, a significant increase in synaptophysin immunolabeled puncta in the hippocampal subregion, CA1, in APP_SWE/PS1_dE9 mice was detected, with no significant synaptic density changes observed in CA3, or the Fr2 region of the prefrontal cortex. Moreover, a significant increase in hippocampal BDNF was detected in APP_SWE/PS1_dE9 mice exposed to EE, however, no changes were detected in neocortex or between Wt animals. These results demonstrate that mid to later‐life cognitive enrichment has the potential to promote synaptic and cognitive health in ageing, and to enhance compensatory capacity for synaptic connectivity in pathological ageing associated with Aβ deposition.     

Advances in the development of biomarkers for Alzheimer’s disease: from CSF total tau and Aβ1–42 proteins to phosphorylated tau protein

Advances have been made to establish biological markers of Alzheimer’s disease (AD). Measurement of total tau (t-tau) and beta-amyloid_1–42 (Aβ_1–42) in the cerebrospinal fluid (CSF) seems useful to discriminate early and incipient AD from age-associated memory-impairment, depression, and some secondary dementias. New immunoassays to detect different phosphorylated tau epitopes (p-tau) have recently been developed. P-tau phosphorylated at threonine 231 (p-tau₂₃₁) showed improvements compared to t-tau in the early detection of AD in subjects with mild cognitive impairment. As p-tau₂₃₁ declined during the course of AD, it may have potential to track disease progression. Additionally, p-tau₂₃₁ improved differential diagnosis between AD, frontotemporal dementia, and geriatric major depression. P-tau phosphorylated at threonine 181 improved diagnostic accuracy between AD and dementia with Lewy bodies. P-tau phosphorylated at serine 199 demonstrated high discriminative power between AD and non-Alzheimer’s dementia. P-tau phosphorylated at serine 306/serine 404 improved differential diagnosis between AD and vascular dementia. A comparative study of the different p-tau epitopes is currently under way.In summary, first clinical multi-center studies suggest that measurement of phosphorylated tau proteins may significantly improve early and differential diagnosis and may come close to fulfilling proposed criteria of a biological marker for AD.     

Sex differences in estrogen receptor α and β expression in vasopressin neurons of the supraoptic nucleus in elderly and Alzheimer’s disease patients: no relationship with cytoskeletal alterations

In various hypothalamic and adjacent brain regions we have previously found a remarkable increase in nuclear estrogen receptor staining in Alzheimer’s disease (AD). In order to see whether this was a general phenomenon or rather specific for those areas that are affected by the AD process we investigated ERα and ERβ expression in the arginine–vasopressin (AVP) neurons of the human dorsolateral suparoptic nucleus (dl-SON), that is the major source of plasma AVP. These neurons remain exceptionally intact in AD. Changes in ER expression were studied in relation to early Alzheimer changes (i.e. hyperphosphorylated tau) and neuronal metabolism in AD as determined by the size of the Golgi apparatus (GA) or cell size. No difference in neuronal metabolism (i.e. GA size or cell size) of AVP neurons was observed between AD and control patients and no early cytoskeletal AD alterations were found confirming the resistance of the dl-SON to AD. While no differences between AD and control patients were present for ERα and ERβ staining except for a lower proportion of nuclear ERβ AVP-positive neurons in AD subjects, complex sex differences not directly related to AD were observed within each group. The main finding of the present study is that in the dl-SON, that remains active and spared of AD changes, the increase in nuclear ERs seen in adjacent affected areas in AD patients does not occur. This indicates that a rise of nuclear ERs is not a generally occurring phenomenon but rather related to the pathogenetic alterations of the AD process.     

The combinations of TNFα–308 and IL‐6 –174 or IL‐10 –1082 genes polymorphisms suggest an association with susceptibility to sporadic late‐onset Alzheimer’s disease

Objective – Single nucleotide polymorphisms in the regulatory regions of the cytokine genes for tumor necrosis factor α (TNFα), interleukin (IL)‐6 and IL‐10 have been suggested to influence the risk of Alzheimer’s disease (AD) with conflicting results. Aim – To investigate the TNFα–308, IL‐6 –174 and IL‐10 –1082 gene polymorphisms as susceptibility factors for AD. Methods – We analyzed genotype and allele distributions of these polymorphisms in 101 sporadic AD patients and 138 healthy controls. Results – Heterozygotes (AG) or combined genotype (AG+AA) for IL‐10 –1082 were associated with approximately two‐fold increase in the risk of AD. Carriers of A alleles of both TNFα–308 and IL‐10 –1082 had 6.5 times higher risk for AD in comparison with non‐carriers. Concomitant presence of both mutant TNFα–308 A and IL‐6 –174 C alleles raised three‐fold the AD risk, whereas there was no notable risk for AD afflicted by IL‐6 –174 polymorphism alone. Conclusion – Our results suggest that TNFα and IL‐10 promoter polymorphism might be a risk factor for AD. The combined effects of TNFα–308, IL‐6 –174 and IL‐10 –1082 variant alleles may be more decisive to induce functional differences and modify the risk for AD.     

Systemic immune‐checkpoint blockade with anti‐PD1 antibodies does not alter cerebral amyloid‐β burden in several amyloid transgenic mouse models

Chronic inflammation represents a central component in the pathogenesis of Alzheimer's disease (AD). Recent work suggests that breaking immune tolerance by Programmed cell Death‐1 (PD1) checkpoint inhibition produces an IFN‐γ‐dependent systemic immune response, with infiltration of the brain by peripheral myeloid cells and neuropathological as well as functional improvements even in mice with advanced amyloid pathology (Baruch et al., ( 2016 ): Nature Medicine, 22:135–137). Immune checkpoint inhibition was therefore suggested as potential treatment for neurodegenerative disorders when activation of the immune system is appropriate. Because a xenogeneic rat antibody (mAb) was used in the study, whether the effect was specific to PD1 target engagement was uncertain. In the present study we examined whether PD1 immunotherapy can lower amyloid‐β pathology in a range of different amyloid transgenic models performed at three pharmaceutical companies with the exact same anti‐PD1 isotype and two mouse chimeric variants. Although PD1 immunotherapy stimulated systemic activation of the peripheral immune system, monocyte‐derived macrophage infiltration into the brain was not detected, and progression of brain amyloid pathology was not altered. Similar negative results of the effect of PD1 immunotherapy on amyloid brain pathology were obtained in two additional models in two separate institutions. These results show that inhibition of PD1 checkpoint signaling by itself is not sufficient to reduce amyloid pathology and that additional factors might have contributed to previously published results (Baruch et al., ( 2016 ): Nature Medicine, 22:135–137). Until such factors are elucidated, animal model data do not support further evaluation of PD1 checkpoint inhibition as a therapeutic modality for Alzheimer's disease.     

Synergistic effect of two oxidative stress‐related genes (heme oxygenase‐1 and GSK3β) on the risk of Parkinson’s disease

Background: Oxidative stress is a central factor in the pathogenesis of Parkinson’s disease (PD). Heme oxygenase‐1 (HO‐1) is an antioxidant protein expressed in response to oxidative challenge, and its expression levels are inversely correlated with glycogen synthase kinase‐3β (GSK3β) activity. Underexpression of HO‐1 in concert with an upregulation of GSK3β would result in a less effective antioxidant response and might increase the risk of PD. Methods: We examined two functional polymorphism in the promoter regions of HO‐1 (?413, rs2071746) and GSK3β (?157, rs6438552) in a group of 251 Spanish patients with PD and 234 controls. Results: Subjects carrying both the HO‐1 (?413, rs2071746) TT genotype and the GSK3β (?157, rs6438552) TT genotype had a four times higher risk of developing PD than subjects without these genotypes (adjusted by age and sex OR = 4.12; 95% CI = 1.45–11.71; Bonferroni corrected P = 0.024). Conclusions: Considering synergistic effects between polymorphisms in oxidative stress‐related genes may help in determining the risk profile for PD.