TGF-β receptors-I and -II immunoexpression in Alzheimer’s disease: a comparison with aging and progressive supranuclear palsy

The transforming growth factor-βs (TGF-βs) influence cell survival, and TGF-β2 shows increased immunoexpression in neurofibrillary tangle-bearing neurons and reactive glia in Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP). We compared immunohistochemical expression of TGF-β type I (RI) and type II (RII) receptors in eight patients with AD, eight controls and three cases of progressive supranuclear palsy. Mild intraneuronal immunoreactivity for the RI receptor was observed in all cases. Intraneuronal TGF-β RII receptor immunoexpression was more common in all groups, and its frequency did not differ between groups. We observed increased immunoreactivity for both RI and RII subtypes in reactive glia in the AD frontal cortex (RI: U = 0.5, p = 0.002; and RII: U = 9.000, p = 0.006) and parahippocampal gyrus (RI: U = 9.500, p = 0.013; RII: U = 14.5, p = 0.05) compared to control cases. We conclude that TGF-β RI and II immunoreactivity is increased in reactive glia in AD and progressive supranuclear palsy, and RI immunoreactivity may occasionally be increased in neurons in cases with advanced AD.     

Progression of neurofibrillary changes and PHF-τ in end-stage Alzheimer’s disease is different from plaque and cortical microglial pathology

In terminal Alzheimer’s disease (AD) the frequency of plaques was found to be reduced in single cases. To test this finding in a larger sample, and in order to determine whether the number of plaques labeled with different markers and the distribution of neurofibrillary tangles are correlated positively to each other and to the degree of dementia, a sample of 134 autopsy brains with and 15 without AD-related pathology has been examined. All of the cases were staged according to Braak and Braak. Both the frequency of plaques immunopositive for β-amyloid, amyloid precursor protein, and apolipoprotein E and that of microglial cells in the cortex and in the white matter were determined semiquantitatively. The content and distribution of PHF-τ was ascertained by ELISA and immunohistochemistry. Both the clinical dementia rating and the global deterioration scale were used as clinical parameters retrospectively. Correlation coefficients were calculated for all parameters and differences were evaluated statistically. With progressive distribution of neurofibrillary tangles and increasing content of PHF-τ, the plaque stages and the degree of cortical microglia reaction increased up to the Braak-stages IV and V, thereafter showing a slightly decreasing tendency in the investigated regions. In end-stage AD resorption of β-amyloid seems to surpass its deposition. The microglial reaction in the white matter correlated neither with the Braak-stage nor with the accumulation of amyloid. With regard to the degree of dementia, both scales correlated well with the pathological changes. Our data show that neuronal cytoskeletal alterations progressively increase with progressive dementia until the end stage of AD in contrast to the frequencies of plaques and cortical microglial cells, and are therefore preferable for staging purposes.     

Inflammatory factors are elevated in brain microvessels in Alzheimer’s disease

In Alzheimer’s disease (AD) inflammatory processes occur in pathologically vulnerable brain regions. The objective of this study is to compare both the release and the presence of microvessel-associated cytokines in vessels isolated from the brains of AD patients to microvessels from control brains. Microvessels are isolated from the cortices of AD patients and age-matched controls, without evidence of neurodegenerative disease. Inflammatory factors in the media are quantitated by ELISA and microvessel-associated mediators assessed by Western blot. Our results demonstrate that unstimulated AD microvessels release significantly higher levels of interleukin-1β-(IL-1β), IL-6, and tumor necrosis factor (TNF-) compared to non-AD microvessels. Levels of microvessel-associated monocyte chemoattractant protein (MCP-1) and IL-1β are high in AD-derived microvessels, but not detectable in non-AD microvessels. These results suggest that the cerebral microcirculation contributes inflammatory mediators to the milieu of the AD brain and may be involved in the pathogenesis of neuronal injury and death in this disorder.     

Downregulation of neuronal cyclooxygenase-2 expression in end stage Alzheimer’s disease

Nonsteroidal anti-inflammatory drugs (NSAIDs) appear to delay the onset of Alzheimer’s disease (AD). NSAIDs inhibit cyclooxygenase (COX), of which two isoforms exist. We report decreased neuronal COX-2 expression in AD subjects relative to nondemented controls using qualitative analysis of COX-2 immunoreactivity and quantification of COX-2 positive neurons in different hippocampal subfields. These changes also occurred in subjects with other dementia and thus may not be disease specific. The proportion of COX-2 positive neurons decreased in subjects with clinical dementia rating (CDR) 5 but not CDR 4, suggesting that this was a late event in the course of the disease. Furthermore, COX-2 was not preferentially associated with paired helical filament immunoreactivity, a marker of neuronal pathology. COX-2 immunoreactivity was also observed in astrocytes and cerebrovasculature. Indeed, the density of COX-2 immunopositive astrocytes was increased in AD temporal cortex. Based on our findings, it is unlikely that neuronal COX-2 contributes to pathology in end stage AD; however, COX-2 in other cell types may participate in the inflammation-related response associated with the disease.     

Abnormalities in Alzheimer’s Disease Fibroblasts Bearing the APP670/671 Mutation

Abnormalities in cultured fibroblasts from familial Alzheimer’s Disease (FAD) cases uniquely enable the determination of how gene defects alter cell biology in living tissue from affected individuals. The current study focused on measures of calcium regulation and oxidative metabolism in fibroblast lines from controls and FAD individuals with the Swedish APP670/671 mutation. Bombesin-induced elevations in calcium in APP670/671 mutation-bearing lines were reduced by 40% (p < 0.05), a striking contrast to the 100% increase seen in sporadic AD and presenilin-1 (PS1) mutation-bearing cells in previously published studies. The APP670/671 mutation-bearing lines did not exhibit the exaggerated 4-bromo-A23187 releasable pool of calcium following 10 nM bradykinin, the enhanced sensitivity of calcium stores to low concentrations of bradykinin, nor the reduced activity of α-ketoglutarate dehydrogenase previously reported in cells from sporadic AD and mutant PS1 FAD. Thus, an altered regulation of internal calcium stores is common to all AD lines, but the calcium pool affected and the polarity of the alteration varies, apparently in association with particular gene mutations. Comparison of signal transduction in cell lines from multiple, genetically characterized AD families will allow testing of the hypothesis that these various pathogenic FAD abnormalities that lead to AD converge at the level of abnormal signal transduction.     

Cell cycle regulatory failure in neurones: causes and consequences

The number of Alzheimer’s disease sufferers shows an alarming increase throughout the world. Therefore elucidation of the pathogenic mechanisms leading to Alzheimer’s disease and the design of effective treatment, preventive or curative, became imperative.

In the last few years several groups have found evidence indicating that the development of Alzheimer-type pathology and the associated excess cell death is the consequence of an aberrant re-entry of neurones into the cell division cycle. We believe that neuronal cell cycle re-entry is followed by regulatory failure that allows neurones to progress into the late stages of the cycle. At this stage, in apoptosis incompetent neurones, the active kinases lead to tau hyperphosphorylation, and the amyloid precursor protein is processed into amyloidogenic fragments. Thus the cell cycle arrest will lead to either the development of Alzheimer’s type pathology or to apoptotic neuronal death.

Although there are several studies aimed at the elucidation of the precise pathways and mechanisms by which the cell cycle disturbances may lead to Alzheimer’s disease there is precious little known about the possible causes of the neuronal cell cycle re-entry. On the other hand we can only speculate on the mechanisms that lead to the subsequent regulatory failure.     

Pro-inflammatory complement activation by the Aβ peptide of Alzheimer’s disease is biologically significant and can be blocked by vaccinia virus complement control protein

The amyloid plaque is the hallmark of Alzheimer’s disease (AD). The transmembrane domain and a portion of the C-terminus (Aβ) of the amyloid precursor protein, are known to form the nucleus of the amyloid plaque. It has been demonstrated recently, using in vitro assays, that the Aβ peptide can activate both the classical (antibody-independent) and alternate pathways of complement activation. The proposed complement activation is due to the binding of Aβ to the complement components Clq and C3, respectively, which initiate formation of the proinflammatory C5a and C5b-9 membrane attack complex. In this report, we have investigated the in vitro findings for the likely complement-dependent proinflammatory properties of the Alzheimer’s disease Aβ peptide. We have performed experiments using congenic C5-deficient and C5-sufficient mice injected with synthetic Aβ and recombinant polypeptide (C-100) containing Aβ. Injection of C-100 into C5-sufficient mice induced a clear increase in the number of polymorphonuclear cells (neutrophils) at the site of injection due to complement activation and the subsequent release of proinflammatory chemtoactic factors. In sharp contrast, the C5-deficient mice did not show any increase in cellular influx. The vaccinia virus complement control protein, an inhibitor of both the classical and alternate pathway can down-regulate the biologically significant activation of complement by Aβ, as demonstrated by an in vitro immunassay. The therapeutic down-regulation of Aβ-caused complement activation could greatly alleviate the progression of some of the chronic neurodegeneration characteristic of Alzheimer’s disease.     

Nogo-A及其受体在阿尔茨海默病中的作用

Nogo-A是一种重要的髓鞘相关生长抑制因子,在成体动物中枢神经系统损伤再生中发挥了关键的抑制作用。近年来许多研究表明, Nogo-A及其受体NgR可以影响淀粉样蛋白Aβ的代谢,其下游的ROCK激酶可以影响Aβ、tau蛋白的水平以及血脑屏障的通透性,这些均提示Nogo-A及其受体与阿尔茨海默病（AD）的发病机制有着密切的联系。此外,最近的研究还发现了Nogo-A的另外两种受体PirB和S1PR2,可能为Nogo-A在神经系统疾病中的作用提供新的研究方向。我们将着重阐释Nogo-A及其受体的基本结构和功能的新发现,以及其在AD中的作用等方面的最新研究进展。     

Anti-inflammatory actions of peroxisome proliferator-activated receptor gamma agonists in Alzheimer’s disease

The role of inflammatory processes in the brains of Alzheimer’s Disease (AD) patients has recently attracted considerable interest. Indeed, the only demonstrated effective therapy for AD patients is long-term treatment with non-steroidal anti-inflammatory drugs (NSAIDs). The mechanistic basis of the efficacy of NSAIDs in AD remains unclear. However, the recent recognition that NSAIDs can bind to and activate the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), has offered an explanation for the action of these drugs in AD. PPARγ activation leads to the inhibition of microglial activation and the expression of a broad range of proinflammatory molecules. The newly appreciated anti-inflammatory actions of PPARγ agonists may allow novel therapies for AD and other CNS indications with an inflammatory component.     

Distribution of VGF peptides in the human cortex and their selective changes in Parkinson's and Alzheimer's diseases

VGF mRNA and its precursor-derived products are selectively expressed in certain neurons and promptly respond to neurotrophins and to neural/electrical activity. Proteomic studies have previously revealed a reduction in some VGF peptides in the cerebrospinal fluid of patients affected by Alzheimer''s disease and other conditions, suggesting their potential diagnostic and clinical significance. As the presence of VGF peptides within the human cortex has been somewhat elucidated, they were studied postmortem in the frontal, parietal, and temporal cortex areas of control subjects and patients affected by Parkinson''s disease, and in parietal cortex samples from patients with Alzheimer''s disease. We raised antibodies to the C-/N-terminal portions of the proVGF precursor protein, to the TPGH and TLQP sequences and to the neuroendocrine regulatory peptide (NERP)-1, all used for enzyme-linked immunosorbent assay coupled with gel chromatography and for immunohistochemistry. In the control brain samples, the levels of TPGH and C-terminus peptides were about 130–200 and 700–2000 pmol g⁻¹, respectively, the N-terminus and NERP-1 peptides were less represented (about 10–30 and 4–20 pmol g⁻¹, respectively), and the TLQP peptides were below detection limits. Upon gel chromatography, the VGF antisera mainly revealed small molecular weight forms (i.e. about 0.8–1.3 kDa), whereas VGF immunolocalisation was found within different types of neuron in rat and bovine brain cortices. In the Parkinson''s disease samples, a clear-cut decrease was revealed in the parietal cortex only, exclusively for TPGH and NERP-1 peptides, whereas in the Alzheimer''s disease samples, a reduction in all of the VGF peptides was shown. The results suggest the involvement of VGF in the physiological or pathophysiological mechanisms occurring in the parietal cortex of patients with Parkinson''s and Alzheimer''s diseases.     

GSK-3 dependent phosphoepitopes recognized by PHF-1 and AT-8 antibodies are present in different tau isoforms

It is widely known that the tau protein that forms the aggregates found in tauopathies like Alzheimer’s disease (AD) is hyperphosphorylated. Many of the sites that are hyperphosphorylated in AD can also be found phosphorylated in non-pathological control brains, although to a lesser extend. Among the different kinases that are able to phosphorylate tau in these sites, GSK-3 has emerged as a key effector of AD pathogenesis in view of its interaction with many of the proteins involved in the ethiology of AD. In this work, we have tested if control samples show only a decrease in the amount of phosphorylated tau molecules, or if the phosphorylation at different sites occurs in different tau isoforms, whereas in the pathological situation a single tau isoform is modified simultaneously at the different sites. Our results indicate that the second possibility takes place and that the differences in the phosphorylation of different tau isoforms could be due to a different subcellular distribution of these different tau isoforms in a neuron.     

Addressing the challenges of transforming laboratory advances into Alzheimer's Disease treatments

This essay addresses the challenges of clinical trials to develop treatments for Alzheimer’s Disease (AD). The issues covered are enrolling subjects, defining clinically meaningful endpoints, and making the claim that a drug slows the progression of the disease. The perspective to address these challenges is that dementia research should embrace a biopsychosocial model for drug development. In this model, the patient and caregiver are seen as interrelated subjects of both treatment and research and outcome measures reflect biomarkers of the disease, the functional morbidity of AD and the distress of caregiving.     

Valproic acid induces caspase 3-mediated apoptosis in microglial cells

Valproic acid is widely used for the treatment of epilepsy and mood disorders, but its mode of action is unclear. Treatment of neuronal cells with valproic acid promotes neurite sprouting, is neuroprotective and drives neurogenesis; however its effects on non-neuronal brain cells are less clear. We report that valproic acid induces apoptosis in the mouse microglial cell line, BV-2, at concentrations within the therapeutic range. When BV-2 cells were incubated for 24 h with 500–1000 μM valproic acid we observed a reduction in cell number, the appearance of apoptotic morphology and increased caspase 3 cleavage. Exposure of a macrophage cell line (RAW 264.7) to similar concentrations of valproic acid also led to reduced cell number but no caspase 3 cleavage, suggesting these cells responded to valproic acid with reduced proliferation rather than apoptosis. This was confirmed using bromodeoxyuridine incorporation studies. Similar concentrations of valproic acid added to Neuro-2a, SK-N-SH and C6 cell lines as well as human NTera-2 astrocytes did not evoke cell death. The caspase 3 inhibitor DEVD-CHO inhibited valproic acid-induced apoptosis in BV-2 cells whereas the MEK inhibitor U0126 potentiated valproic acid-mediated apoptosis. These results demonstrate that valproic acid selectively induces apoptosis in BV-2 cells by way of a caspase 3-mediated action. As activated microglia secrete neurotoxins in neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and HIV dementia, valproic acid may alleviate these diseases by selectively killing microglia.     

Clogging of axons by tau, inhibition of axonal traffic and starvation of synapses

Loss of synapses and dying back of axons are considered early events in brain degeneration during Alzheimer’s disease. This is accompanied by an aberrant behavior of the microtubule-associated protein tau (hyperphosphorylation, aggregation). Since microtubules are the tracks for axonal transport, we are testing the hypothesis that tau plays a role in the malfunctioning of transport. Experiments with various neuronal and non-neuronal cells show that tau is capable of reducing net anterograde transport of vesicles and cell organelles by blocking the microtubule tracks. Thus, a misregulation of tau could cause the starvation of synapses and enhanced oxidative stress, long before tau detaches from microtubules and aggregates into Alzheimer neurofibrillary tangles. In particular, the transport of amyloid precursor protein is retarded when tau is elevated, suggesting a possible link between the two key proteins that show abnormal behavior in Alzheimer’s disease.     

龟龄集对 AD 小鼠皮层和海马 Fas/ FasL 表达及神经元凋亡的影响

目的 探究龟龄集对阿尔茨海默病 (Alzheimer’s disease, AD) 小鼠皮层和海马 Fas/ FasL 表达及 神经元凋亡的影响。 方法 构建 AD 小鼠模型, 小鼠随机分为对照组、 模型组、 多奈哌齐组及龟龄集低、 中、 高剂量组。 Morris 水迷宫检测干预前后小鼠的学习记忆能力; HE 染色检测小鼠皮层、 海马神经元病理 学改变情况; TUNEL 染色检测神经元凋亡情况; Western 印迹及 Real time PCR 分别检测 Fas、 FasL 的蛋白 表达水平及 mRNA 表达水平。 结果 与模型组比较, 龟龄集各组和多奈哌齐组小鼠学习记忆能力明显提高 (P< 0. 05), 皮层和海马神经元病理学损害及神经元凋亡改善 (P< 0. 05), Fas、 FasL 蛋白和 mRNA 水平下 降 (P< 0. 05)。 结论 龟龄集可能通过抑制 Fas/ FasL 表达抑制 AD 模型小鼠皮层、 海马神经元凋亡, 并改 善其学习记忆能力。     

Aminopyridazines inhibit beta-amyloid-induced glial activation and neuronal damage in vivo

The critical role of chronic inflammation in disease progression continues to be increasingly appreciated across multiple disease areas, especially in neurodegenerative disorders such as Alzheimer’s disease. We report that late intervention with a recently discovered aminopyridazine suppressor of glial activation, developed to inhibit both oxidative and inflammatory cytokine pathways, attenuates human amyloid beta (Aβ)-induced glial activation in a murine model. Peripheral administration of the aminopyridazine MW01-070C, beginning 3 weeks after the start of intracerebroventricular infusion of human Aβ1-42, decreased the number of activated astrocytes and microglia and the levels of proinflammatory cytokines interleukin-1β, tumor necrosis factor- and S100B in the hippocampus. Inhibition of neuroinflammation correlated with a decreased neuron loss, restoration towards control levels of synaptic dysfunction biomarkers in the hippocampus, and diminished amyloid plaque deposition. The results from this in vivo chemical biology approach provide a proof of concept that targeting of key glia inflammatory cytokine pathways can suppress Aβ-induced neuroinflammation in vivo, with resultant attenuation of neuronal damage.     

Association analysis of genes involved in cholesterol metabolism located within the linkage region on chromosome 10 and Alzheimer's disease

Epidemiological studies identified a higher risk of developing Alzheimer’s disease (AD) among subjects with elevated cholesterol levels. This association may be caused by a modulation of the amyloid precursor protein (APP) processing in response to the cellular cholesterol content. High cholesterol levels may favor the amyloidogenic pathway by inhibition of the -secretase probably leading to elevated beta-Amyloid (Aβ) production. The identification of a linkage peak on chromosome 10q using high Aβ as quantitative trait led us to examine polymorphisms of genes located on chromosome 10 involved in cholesterol metabolism, like Lipase A (LIPA), Cholesterol 25 hydroxylase (CH25H), and FLJ22476, a high density lipoprotein binding related protein. Using 286 patients with AD and 162 controls we analyzed several single nucleotide polymorphisms (SNPs) within LIPA, CH25H, and FLJ22476. None of the polymorphisms showed significant association with AD which contradicts recent findings on CH25H. From our results we conclude that the investigated genetic variations do not contribute to the genetic risk of AD.     

Nimodipine Ameliorates Impaired Eyeblink Classical Conditioning in Older Rabbits in the Long-Delay Paradigm

Research using the hippocampally dependent short-conditioned stimulus trace conditioning paradigm demonstrated that nimodipine ameliorated learning deficits in older rabbits. Evidence from in vitro and in vivo measures indicated that the site of drug action was hippocampal pyramidal cells. Acquisition occurs in the long (750 ms) delay conditioning paradigm in the absence of the hippocampus. This experiment with 40 older rabbits was undertaken to determine if nimodipine ameliorates impaired acquisition in a conditioning paradigm not dependent on the hippocampus. Fifteen 90-trial sessions of paired conditioning stimuli were presented to 3 groups receiving daily injections of 0, 1, or 5 mg/kg nimodipine. Explicitly unpaired control groups received 0 or 5 mg/kg nimodipine. Acquisition with the 5 mg/kg dose was significantly faster. Existing evidence suggested that nimodipine acted in the hippocampus, but we could not rule out the possibility that the drug also affected conditioning via the cerebellum.