Emerging Alzheimer's disease therapies: inhibition of beta-secretase

Among the approaches towards disease modifying treatment of Alzheimer’s disease blocking the initial step of the amyloid cascade, Aβ42 generation, has received most attention. Aβ42 generation requires two proteases, β- and γ-secretase, and inhibition of these enzymes is a key focus of AD drug development. Progress in this area has been slow, because these enzymes were not identified. Using an expression cloning strategy we have identified a novel membrane bound aspartic protease, BACE1, as β-secretase. The enzyme has been characterized in detail. The x-ray crystal structure, which is critical for rational inhibitor design, has been solved and shown to be similar to that of other pepsin family members. Our recent knockout studies show that BACE1 is critical for Aβ generation, but the knockout mice show an otherwise normal phenotype, raising the possibility that therapeutic BACE1 inhibition could be accomplished without major mechanism based toxicity. However, target-mediated toxicity of β-secretase inhibition cannot be ruled out, as long as the major substrates of this enzyme are unknown. While various peptidic β-secretase inhibitors have been published, the key challenge now is the generation of more drug-like compounds that could be developed for therapeutic purposes.     

Neurogenesis in Alzheimer's disease

It is widely acknowledged that neural stem cells generate new neurons through the process of neurogenesis in the adult brain. In mammals, adult neurogenesis occurs in two areas of the CNS: the subventricular zone and the subgranular zone of the dentate gyrus of the hippocampus. The newly generated cells display neuronal morphology, generate action potentials and receive functional synaptic inputs, their properties being equivalent to those of mature neurons. Alzheimer's disease (AD) is the widespread cause of dementia, and is an age-related, progressive and irreversible neurodegenerative disease that results in massive neuronal death and deterioration of cognitive functions. Here, we overview the relations between adult neurogenesis and AD, and try to analyse the controversies in the field. We also summarise recent data obtained in the triple transgenic model of AD that show time- and region-specific impairment of neurogenesis, which may account for the early changes in synaptic plasticity and cognitive impairments that develop prior to gross neurodegenerative alterations and that could underlie new rescue therapies.     

Increased galanin receptor occupancy in Alzheimer's disease

Increased galanin (GAL) may be associated with the cognitive deficits characteristic of Alzheimer’s disease (AD). However, both increased and decreased GAL receptor density has been reported in AD brain. Previous studies indicate pre-treatment with guanine nucleotides displaces endogenous GAL from GAL receptors (GALR), providing an indirect measurement of GALR occupancy. In addition, pre-treatment with guanine nucleotides may provide a more accurate measurement of GALR density since it would avoid the masking of GALRs by residual binding of endogenous GAL. Thus, in the present study, we examined the influence of pre-treatment with guanine nucleotides on ¹²⁵I-GAL binding in multiple regions of normal and AD brain. Our results indicate that GTP pre-treatment enhances GAL binding in specific regions in normal and AD brain. In addition, our results suggest an increase in the number of GALRs occupied by endogenous GAL in the deep layers of the frontal cortex and the lateral hypothalamus of AD subjects compared to normal subjects. The regional differences in GALR density and receptor occupancy between normal and AD subjects may play a role in the cognitive disturbances associated with the disease.     

Cognitive and behavioral heterogeneity in Alzheimer's disease: seeking the neurobiological basis

Alzheimer’s disease (AD) is manifested by core features of progressive memory impairment, visuospatial decline, aphasia, and loss of executive function. In addition, patients may evidence a variety of other cognitive and behavioral features. The neurobiological basis for this clinical heterogeneity is uncertain but corresponding abnormalities on functional imaging suggest that variations in the distribution of the pathogenic changes in AD account for some of the observed clinical differences. Behavioral as well as cognitive variability has been correlated with disturbances on positron emission tomography and single photon emission computerized tomography. Functional imaging can reveal characteristic brain activity changes in AD, distinguish AD from other dementia syndromes, assess the integrity of transmitter systems in AD, determine the effect of cognitive enhancing and psychotropic drugs on metabolism and transmitter system function in AD, and possibly predict treatment responsiveness. Animal models of AD may improve our understanding of clinical variations in human AD. Thus far, development of cognitive tests for transgenic mice with AD pathology has been limited. Evaluations paralleling human neuropsychological tests are needed. In addition, technologies facilitating behavioral observations relevant to psychosis, depression, apathy, and agitation in AD have not been developed for transgenic models. Application of experiments inducing animal equivalents of depression and psychosis to determine the vulnerability of animal models of AD to these conditions may provide additional insights into human neuropsychiatric symptoms in AD. The efficacy of psychotropic drugs can be assessed in animal models of AD subjected to the provocative stimuli used in experimental models of psychopathology. There are a plethora of opportunities for basic scientists to offer insights, develop strategies, and provide techniques and technologies relevant to understanding the clinical manifestations of AD.     

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.     

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.     

Astrocytic cytoskeletal atrophy in the medial prefrontal cortex of a triple transgenic mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the loss of cognitive functions, reflecting pathological damage to the medial prefrontal cortex (mPFC) as well as to the hippocampus and the entorhinal cortex. Astrocytes maintain the internal homeostasis of the CNS and are fundamentally involved in neuropathological processes, including AD. Here, we analysed the astrocytic cytoskeletal changes within the mPFC of a triple transgenic mouse model of AD (3 × Tg-AD) by measuring the surface area and volume of glial fibrillary acidic protein (GFAP)-positive profiles in relation to the build-up and presence of amyloid-β (Aβ), and compared the results with those found in non-transgenic control animals at different ages. 3 × Tg-AD animals showed clear astroglial cytoskeletal atrophy, which appeared at an early age (3 months; 33% and 47% decrease in GFAP-positive surface area and volume, respectively) and remained throughout the disease progression at 9, 12 and 18 months old (29% and 36%; 37% and 35%; 43% and 37%, respectively). This atrophy was independent of Aβ accumulation, as only a few GFAP-positive cells were localized around Aβ aggregates, which suggests no direct relationship with Aβ toxicity. Thus, our results indicate that the progressive reduction in astrocytic branching and domain in the mPFC can account for the integrative dysfunction leading to the cognitive deficits and memory disturbances observed in AD.     

Synaptic pathology in Alzheimer's disease: a review of ultrastructural studies

Morphologic studies of the neuropathology in Alzheimer’s disease (AD) have demonstrated significant loss of synaptic connectivity in many regions of the neocortex and hippocampus. The strongest correlation with cognitive decline in AD is with the synaptic density. This article discusses the ultrastructural studies that have documented changes in synaptic numbers in many areas of association cortex and in the hippocampal dentate gyrus molecular layer. Changes in the synaptic complex are discussed as a possible compensatory mechanism in response to synapse loss and a model is proposed to help relate the significance of these synaptic changes. Comparisons are made between results observed with ultrastructural technique and those utilizing immunohistochemistry to assess changes in synaptic pathology. Possible reasons underlying the synaptic neuropathology are discussed.     

Familial Alzheimer's disease presenilin-1 mutants potentiate cell cycle arrest

We previously reported that overexpression of presenilin 1 and 2 (PS1 and PS2) in HeLa cells leads to cell cycle arrest, and that the PS2(N141I) FAD mutant potentiates cell cycle arrest compared to wild-type PS2. Using similar BrdU incorporation studies we now report that three different PS1 FAD mutants also increase cell cycle arrest compared to wild-type PS1when overexpressed in either HeLa cells or an ATM deficient cell line. We detected reproducible differences in the degrees to which these FAD mutants induced arrest. PS1(P117L) reduced BrdU incorporation the most (13 to 14%) followed by PS1(P267S) (7.5 to 9%), with the PS1(E280A) mutant inhibiting BrdU incorporation the least (6 to 7%), compared to wild-type PS1. The degree to which the different mutants inhibited cell cycle progression correlates somewhat with the age of AD onset induced by the mutations in carriers. Immunoblot analysis of protein extracts from presenilin-overexpressing cells indicates that the cell cycle-regulated cytoplasmic pool of β-catenin is dramatically reduced, whereas the insoluble β-catenin pool remains essentially unaffected. We discuss the implications of these findings in relationship to cell cycle arrest, apoptosis and AD.     

gamma-Secretase: characterization and implication for Alzheimer disease therapy

γ-Secretase is a membrane-bound protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C-termini of the Aβ peptides which are believed to play a central role in the neuropathology of Alzheimer’s disease. An in vitro γ-secretase assay using a recombinant substrate C100Flag has been developed to facilitate the characterization and identification of this enigmatic protease. Biochemical studies establish that γ-secretase activity is catalyzed by a PS1-containing macromolecular complex. Moreover, the fact that the photoreactive active γ-secretase inhibitor directed to the active site labels PS1 suggests that PS1 contains the active site of the protease. Presenilin/γ-secretase as a potential target for AD therapy and its role in regulated intramembrane proteolysis are discussed.     

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.     

Aβ as a bioflocculant: implications for the amyloid hypothesis of Alzheimer’s disease

Research into Alzheimer’s disease (AD) has been guided by the view that deposits of fibrillar amyloid-β peptide (Aβ) are neurotoxic and are largely responsible for the neurodegeneration that accompanies the disease. This ‘amyloid hypothesis’ has claimed support from a wide range of molecular, genetic and animal studies. We critically review these observations and highlight inconsistencies between the predictions of the amyloid hypothesis and the published data. We show that the data provide equal support for a ‘bioflocculant hypothesis’, which posits that Aβ is normally produced to bind neurotoxic solutes (such as metal ions), while the precipitation of Aβ into plaques may be an efficient means of presenting these toxins to phagocytes. We conclude that if the deposition of Aβ represents a physiological response to injury then therapeutic treatments aimed at reducing the availability of Aβ may hasten the disease process and associated cognitive decline in AD.     

Sex differences in prefrontal volume with aging and Alzheimer's disease

We used volumetric magnetic resonance imaging to examine sex differences in prefrontal tissue volumes of healthy aged and patients with Alzheimer’s disease (AD). Healthy subjects had greater total prefrontal volume than AD, and men had greater total prefrontal volume than women (ps ≤ 0.02). This was true for both gray and white matter volumes. There were no interactions between group and sex for total prefrontal volume. An exploratory analysis of each group suggested that sex differences in both gray and white matter in healthy aging are not sustained in AD.     

血清铜/锌比值对何杰金淋巴瘤分型及预后的评价

目的:探讨血清铜/锌比值在何杰金淋巴瘤(HD)的病理分型及其预后评价的作用。方法:采用原子吸收分光光度计对135例HD患者血清铜水平(SCL),锌水平(SZL)及铜/锌比值(CZR)进行测定。结果:测定结果表明,HD患者SCL和CZR明显高于健康对照组,而SZL显著低于健康对照组,P值均小于0.01。同时在HD患者中,淋巴细胞消减型(LD)患者组SCL和CZR明显高于混合细胞型(MC)组,而MC组则显著高于结节硬化型(NS),NS组又显著高于淋巴细胞为主型(LP),均P＜0.01。并且呈高度的线性关系,而SZL逐渐降低,但线性关系不太明显。结果还表明,HD患者的病情发展时,SCL和CZR明显上升,而SZL下降,当治疗有效,病情好转时,SCL和CZR下降,SZL上升,当病情获得完全缓解时,SCL和CZR趋于正常,而复发时则又升高。结论:HD患者SCL,特别是CZR对HD的病理分型、预后观测,病因探讨有重要的参考价值。     

Amyloid-like inclusions in Huntington's disease

Huntington's disease is a progressive, autosomal dominantly inherited, neurodegenerative disease that is characterized by involuntary movements (chorea), cognitive decline and psychiatric manifestations. This is one of a number of late-onset neurodegenerative disorders caused by expanded glutamine repeats, with a likely similar biochemical basis. Immunohistochemical studies on Huntington's disease tissue, using antibodies raised to the N-terminal region of huntingtin (adjacent to the repeat) and ubiquitin, have recently identified neuronal inclusions within densely stained neuronal nuclei, peri-nuclear and within dystrophic neuritic processes. However, the functional significance of inclusions is unknown. It has been suggested that the disease-causing mechanism in Huntington's disease (and the other polyglutamine disorders) is the ability of polyglutamine to undergo a conformational change that can lead to the formation of very stable anti-parallel beta-sheets; more specifically, amyloid structures. We examined, using Congo Red staining and both polarizing and confocal microscopy, post mortem human brain tissue from five Huntington's disease cases, two Alzheimer's disease cases and two normal controls. Brains from five transgenic mice (R6/2)(12) expressing exon 1 of the human huntingtin gene with expanded polyglutamine, and five littermate controls, were also examined by the same techniques. We have shown that some inclusions in Huntington's disease brain tissue possess an amyloid-like structure, suggesting parallels with other amyloid-associated diseases such as Alzheimer's and prion diseases.     

A sex difference and no effect of ApoE type on the amount of cytoskeletal alterations in the nucleus basalis of Meynert in Alzheimer's disease

In the nucleus basalis of Meynert (NBM) we studied the presence of early cytoskeletal alterations as shown by the antibody Alz-50 in ApoE-typed patients. Using an image analysis system, the area covered by Alz-50 staining and the percentage of neurons stained by Alz-50 were determined. There were no significant differences in the area covered by Alz-50 or in the proportion of Alz-50-stained neurons in the nucleus basalis of Meynert of Alzheimer’s disease (AD) patients with one or two ApoE ε4 alleles as compared with those without any ApoE e4 allele. However, there was a significant sex difference in Alz-50 staining: female Alzheimer’s disease patients showed more severe early cytoskeletal alterations than males. We also found a significant relationship between the number of Alz-50-stained neurons and the severity of dementia.     

Inflammasomes as therapeutic targets for Alzheimer's disease

Alzheimer's disease is the most common form of progressive dementia, typified initially by short term memory deficits which develop into a dramatic global cognitive decline. The classical hall marks of Alzheimer's disease include the accumulation of amyloid oligomers and fibrils, and the intracellular formation of neurofibrillary tangles of hyperphosphorylated tau. It is now clear that inflammation also plays a central role in the pathogenesis of the disease through a number of neurotoxic mechanisms. Microglia are the key immune regulators of the CNS which detect amyloidopathy through cell surface and cytosolic pattern recognition receptors (PRRs) and respond by initiating inflammation through the secretion of cytokines such as interleukin‐1β (IL‐1β). Inflammasomes, which regulate IL‐1β release, are formed following activation of cytosolic PRRs, and using genetic and pharmacological approaches, NLRP3 and NLRP1 inflammasomes have been found to be integral in pathogenic neuroinflammation in animal models of Alzheimer's disease. Therefore, the inflammasomes are very promising novel pharmacological targets which merit further research in the continued endeavor for efficacious therapeutics for Alzheimer's disease.