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Protein aggregates and dementia: Is there a common toxicity?
Abstract
This review considers some of the recent advances made in the understanding of the pathogenic proteins known to aggregate and be implicated in neurodegenerative dementing disorders. It concentrates on the two most obvious candidates for the role of toxic protein in Alzheimer's disease (AD)--beta-amyloid peptide and tau--but also considers other proteins in this disorder and in less common but equally devastating diseases.
... The tandem repeats (3 or 4) are shown in white bars. The number of aminoacids is indicated on the right-hand side (modified from Spillantini and Goedert, 2000) humans and hence is likely to be a pathogenic protein (Lovestone and McLoughlin, 2002). ...
... The major candidate tau kinases include mitogen-activated protein kinase, glycogen synthase kinase 3β, cyclin-dependent kinase 2 (cdk2), cyclin-dependent kinase 5, cAMP-dependent protein kinase, Ca 2+ /calmodulin-dependent protein kinase II, microtubule-affinity regulating kinase, and stress-activated protein kinases (Trojanowski and Lee, 2002). The available evidence points to glycogen synthase kinase-3 being the predominant tau kinase in the brain, although other kinases also phosphorylate tau (Lovestone and McLoughlin, 2002). Protein phosphatases counterbalance the effects of tau kinases, although their role in vivo is unclear. ...
... All these diseases have in common the presence of aberrant tau aggregates. Tau was first implicated in the pathogenesis of Alzheimer's disease when it was discovered to be a major component of the neurofibrillary tangle (Lovestone and McLoughlin, 2002). Subsequently the occurrence of neurofibrillary tangles in a wide range of conditions led to the suggestion that tau deposition may be an incidental nonspecific finding associated with cell death or cellular dysfunction. ...
Tau protein is a neuronal microtubule associated protein, which localizes primarily in the axon. It plays a major role in promoting microtubule assembly, stabilizing microtubules and maintaining the normal morphology of the neurons. Structurally tau is a heterogenous molecule due to several posttranslational modifications. Tauopathies are a group of disorders that are the consequence of abnormal tau phosphorylation, abnormal levels of tau, abnormal tau splicing, or mutations in the tau gene. These disorders are characterized not only by neuronal, but also oligodendroglial and astrocytic filamentous tau inclusions. Tauopathies are the commonest among the neurodegerative diseases with filamentous inclusions. Tauopathies include frontotemporal dementia, Parkinsonism plus syndromes, neuromuscular disorders, and certain genetic and metabolic syndromes. The occurrence of neurofibrillary tangles in a wide range of conditions, including Alzheimer’s disease, initially led to the suggestion that tau deposition may be an incidental nonspecific finding associated with cell death or cellular dysfunction. Later the discovery of close to 20 different mutations in tau in frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17) clearly showed that dysfunction of tau protein causes neurodegeneration and dementia. Among the tauopathies, the most studied is Alzheimer’s disease. Frontotemporal dementia, progressive supranuclear palsy, and corticobasal ganglionic degeneration are some of the other common tauopathies that have been extensively studied. Overlap of clinical and histopathological features occurs between various tauopathies. The role of CSF tau in the diagnosis of dementias is under investigation. The measures of total tau as well as species of phospho-tau detected by antibodies in CSF correlates best with a diagnosis of AD. The discovery of a tau transgenic mouse model has paved the way for testing various therapeutic models for targeting tau.
... 22 Una mutación en el cromosoma 14, y específicamente en el gen de la presenilina 1 (ps-1), se caracteriza por el comienzo de los síntomas alrededor de los 54 años con una duración promedio de 6-7 años. 23 A nivel del cromosoma 1, el gen de la preselinina 2 (ps-2) codifica para la proteína STM2 del citoplasma de las neuronas, en estos casos las manifestaciones clínicas de la enfermedad comienzan alrededor de los 53 años con una duración promedio de 11 años. 21 Se han reportado alteraciones en el cromosoma 12, así como también, la más frecuente de todas, en el cromosoma 19, donde se encuentra el gen de la apolipoproteína E (Apo E), implicado tanto en las formas familiares de comienzo tardío, como en el Alzheimer esporádico. ...
... 21 Se han reportado alteraciones en el cromosoma 12, así como también, la más frecuente de todas, en el cromosoma 19, donde se encuentra el gen de la apolipoproteína E (Apo E), implicado tanto en las formas familiares de comienzo tardío, como en el Alzheimer esporádico. 23 Estos datos configuran a la EA como una enfermedad cuyo origen puede deberse a mutaciones de distintos genes, entre los que se encuentran los que codifican para la Apo E y la cistatina C. 24 La posesión, por herencia, del gen Apo E-4, en el cromosoma 19 parece ser uno de los factores mejor caracterizados para posibilitar la aparición de la EA. La Apo E es una proteína plasmática implicada en el transporte del colesterol y otros lípidos en los diferentes tejidos. ...
RESUMEN La enfermedad de Alzheimer es neurodegenerativa, progresiva, reconocida como un problema creciente en el orden médico, epidemiológico, sociológico y económico. Afecta aproximadamente al 10 % de la población mayor de 65 años y al 40 % en grupos de 80 años o más. En la presente revisión se abordan aspectos relacionados con su epidemiología, factores de riesgo, mecanismos celulares y moleculares. Estos últimos revelan que la formación de betaamilode y otros derivados de la proteína precursora de amiloide son los principales responsables de los cambios en el cerebro de pacientes con Alzheimer. Se incluyeron además la disfunción mitocondrial y de neurotransmisores, el estrés oxidativo, la inflamación, los trastornos neuroinmunes y tróficos. El conocimiento de estas alteraciones permite dilucidar nuevos blancos terapéuticos.
... Low muscle mass consequent to a negative net protein balance could also reflect lower protein concentrations in the brain, indirectly affecting cognition. Abnormal depositions of misfolded and aggregated proteins are common in several types of dementia (84,85). Due to oxidative damage, several proteins as markers of oxidative stress accumulate. ...
Low skeletal muscle mass is associated with cognitive impairment and dementia in older adults. This review describes the possible underlying pathophysiological mechanisms: systemic inflammation, insulin metabolism, protein metabolism and mitochondrial function. We hypothesize that the central tenet in this pathophysiology is the dysfunctional myokine secretion consequent to minimal physical activity. Myokines, such as fibronectin type III domain containing 5/irisin and cathepsin B are released by physically active muscle and cross the blood-brain-barrier. These myokines upregulate local neurotrophin expression such as brain-derived neurotrophic factor (BDNF) in the brain microenvironment. BDNF exerts anti-inflammatory effects that may be responsible for neuroprotection. Altered myokine secretion due to physical inactivity exacerbates inflammation and impairs muscle glucose metabolism, potentially affecting the transport of insulin across the blood-brain barrier. Our working model also suggests other underlying mechanisms. A negative systemic protein balance, commonly observed in older adults, contributes to low skeletal muscle mass and may also reflect deficient protein metabolism in brain tissues. As a result of age-related loss in skeletal muscle mass, decrease in the abundance of mitochondria and detriments in their function, lead to a decrease in tissue oxidative capacity. Dysfunctional mitochondria in skeletal muscle and brain result in the excessive production of reactive oxygen species, which drives tissue oxidative stress and further perpetuates the dysfunction in mitochondria. Both oxidative stress and accumulation of mitochondrial DNA mutations due to aging drive cellular senescence. A targeted approach in the pathophysiology of low muscle mass and cognition could be to restore myokine balance by physical activity.
... Alzheimer's disease (AD) is the primary cause of progressive dementia, followed by Lewy body dementia (LBD) and frontotemporal dementia (FTD) (Sancesario and Bernardini, 2018). A common, pathological hallmark of these dementias is the accumulation of misfolded proteins into insoluble proteinaceous deposits in and around affected tissues (Lovestone and McLoughlin, 2002). In most cases, the major constituent of an insoluble aggregate is a disease-specific protein, such as amyloid-β (Aβ) in AD, α-synuclein (α-syn) in LBD, or TAR DNA-binding protein 43 (TDP-43) in FTD. ...
A number of neurodegenerative diseases are associated with the accumulation of misfolded proteins, including Alzheimer’s disease (AD). In AD, misfolded proteins such as tau and amyloid-β (Aβ) form pathological insoluble deposits. It is hypothesized that molecules capable of dissolving such protein aggregates might reverse disease progression and improve the lives of afflicted AD patients. Here we report new functions of the highly conserved mammalian protein, Fas Apoptosis Inhibitory Molecule (FAIM). We found that FAIM-deficient Neuro 2A cells accumulate Aβ oligomers/fibrils. We further found that recombinant human FAIM prevents the generation of pathologic Aβ oligomers and fibrils in a cell-free system, suggesting that FAIM functions without any additional cellular components. More importantly, recombinant human FAIM disaggregates and solubilizes established Aβ fibrils. Our results identify a previously unknown, completely novel candidate for understanding and treating irremediable, irreversible, and unrelenting neurodegenerative diseases.
... Broad evidence from laboratory, preclinical and clinical studies suggests that amyloid-beta aggregation can also drive the progression of tauopathy (reviewed by Stancu et al., 2014;Hanseeuw et al., 2019), which is increasingly believed to play a key role in cognitive decline (Nelson et al., 2012;Di et al., 2016;Digma et al., 2019). The precise, mechanistic roles of these proteopathies in the aetiology and pathogenesis of AD are the subject of extensive research and debate; however, pathogenic protein aggregation remains strongly implicated in the disease (Lovestone and McLoughlin, 2002;Thal and Fändrich, 2015;Jouanne et al., 2017;Gandhi et al., 2019;Johnson et al., 2019). We will briefly outline the characteristics of amyloid-beta and tau and their aggregation pathways. ...
A substantial body of evidence indicates cationic, arginine-rich peptides (CARPs) are effective therapeutic compounds for a range of neurodegenerative pathologies, with beneficial effects including the reduction of excitotoxic cell death and mitochondrial dysfunction. CARPs, therefore, represent an emergent class of promising neurotherapeutics with multimodal mechanisms of action. Arginine itself is a known chaotrope, able to prevent misfolding and aggregation of proteins. The putative role of proteopathies in chronic neurodegenerative diseases such as Alzheimer’s disease (AD) warrants investigation into whether CARPs could also prevent the aggregation and cytotoxicity of amyloidogenic proteins, particularly amyloid-beta and tau. While monomeric arginine is well-established as an inhibitor of protein aggregation in solution, no studies have comprehensively discussed the anti-aggregatory properties of arginine and CARPs on proteins associated with neurodegenerative disease. Here, we review the structural, physicochemical, and self-associative properties of arginine and the guanidinium moiety, to explore the mechanisms underlying the modulation of protein aggregation by monomeric and multimeric arginine molecules. Arginine-rich peptide-based inhibitors of amyloid-beta and tau aggregation are discussed, as well as further modulatory roles which could reduce proteopathic cytotoxicity, in the context of therapeutic development for AD.
... Whether or not the same PRPs are responsible for different types (e.g., LTP versus LTD), aspects (e.g., functional versus structural) or phases (e.g., consolidation versus reconsolidation) of the plasticity process is an area of ongoing study. 3. Aggregating proteins as solid-state synaptic tags A striking commonality between the most common age-related dementias is the presence of so-called misfolded and aggregated proteins [120]. Currently, aggregated proteins such as Aβ, α-synuclein (α-syn), Huntingtin, TAR DNA binding protein (TDP-43), superoxide dismutase 1 (SOD1), transthyretin, prion protein and tau are associated with over 30 different neurodegenerative diseases. ...
Alzheimer’s Disease is defined as progressive memory loss coincident with accumulation of aggregated amyloid beta and phosphorylated tau. Identifying the relationship between these features has guided Alzheimer’s Disease research for decades, principally with the view that aggregated proteins drive a neurodegenerative process. Here I propose that amyloid beta and phospho-tau write-protect and tag neuroplastic changes as they form, protecting and insuring established neuroplasticity from corruption. In way of illustration, binding of oligomeric amyloid beta to the prion receptor is presented as an example possible mechanism. The write-protecting process is conjected to occur at least partially under the governance of isodendritic neuromodulators such as norepinephrine and acetylcholine. Coincident with aging, animals are exposed to accumulating amounts of memorable information. Compounded with recent increases in life expectancy and exposure to information-rich environments this causes aggregating proteins to reach unforeseen toxic levels as mnemonic circuits overload. As the brain cannot purposefully delete memories nor protect against overaccumulation of aggregating proteins, the result is catastrophic breakdown on cellular and network levels causing memory loss.
... Such a sheet-rich tautomeric isomer can be highly toxic because toxicity is assumed to emerge from the enhanced β-sheet formation in tau. 51,52 In this tautomeric isomer, β-structures mainly occur close to the head region (K8−N13), near the middle portion (K32−H33), and adjacent to the tail region (V40−Y44). In particular, βdominating formation in two regions, namely K8−N13 and V40−Y44, were more pivotal for tau accumulation and formation of NFTs. ...
Histidine tautomerism is considered a crucial component that affects the constitutional and accumulation characteristics of the tau267-312 monomer in the neutral condition, which are connected with the pathobiology of Alzheimer's disease (AD). Interpreting the organizational characteristics and accumulation procedure is a challenging task because two tautomeric conformations (the Nϵ-H or Nδ-H tautomer) can occur in the open neutral condition. In the current work, replica-exchange molecular dynamics (REMD) simulations were performed to investigate the structural properties of the tau267-312 monomer considering the histidine tautomeric effect. Based on the simulation outcomes, the histidine 268 (H268) (δ)-H299 (δ) (δδ) isomer had the highest β-sheet content with a value of 26.2%, which acquires a sheet-governing toxic conformer with the first abundant conformational state of 22.6%. In addition, δδdisplayed notable antiparallel β-sheets between lysine 8 (K8)-asparagine 13 (N13) and valine 40 (V40)-tyrosine 44 (Y44) as well as between K32-H33 and V40-Y44 (β-meander supersecondary structure), indicating this tautomeric isomer may exist to stimulate tau oligomerization. Furthermore, H299 was found to play an essential role in the structural stabilization of the δδisomer compared with H268. The present research will aid in obtaining insight into the organizational and accumulation properties of tau protein in the presence of histidine tautomerism to control AD.
... In addition, it is assumed that the structure and toxicity of tau may be influenced by the Ab peptide. Tau aggregates cause cytotoxicity, and the precise mechanism of the induced toxicity remains to be determined (81). The toxic behaviour of tau in AD appears to be solely due to its abnormal hyperphosphorylation, because dephosphorylation of pathological tau changed it to a normal protein (82). ...
Amyloid-β (Aβ) peptide and tau protein are two hallmark proteins in Alzheimer's disease (AD), however the parameters which mediate the abnormal aggregation of Aβ and tau have not been fully discovered. Here, we have provided an optimum method to purify tau protein isoform 1N4R by using Ni-NTA agarose chromatography under denaturing condition. The biochemical and biophysical properties of the purified protein was further characterized using in vitro tau filament assembly, tubulin polymerization assay, circular dichroism (CD) spectroscopy and atomic force microscopy. Afterwards, we investigated the effect of tau protein on aggregation of Aβ (25-35) peptide using microscopic imaging and cell viability assay. Incubation of tau at physiologic and supra-physiologic concentrations with Aβ25-35 for forty days under reducing and non-reducing conditions revealed formation of two types of aggregates with distinct morphologies and dimensions. In non-reducing condition, the co-incubated sample showed granular aggregates, while in reducing condition, they formed annular protofibrils. Results from cell viability assay revealed the increased cell viability for the co-incubated sample. Therefore, the disassembling action shown by tau protein on Aβ25-35 suggests the possibility that tau may have a protective role in preventing Aβ peptide from acquiring the cytotoxic, aggregated form against oxidative stress damages.
... Through overexpression of the wild-type and deleted forms of CRYBA1 cDNA plasmids, we found that the protein level of CRYBA1 was evidently decreased by the p.G91del mutation, as presented in Figs. 4 and 5, suggesting that this one amino acid deletion could decrease the protein stability and might consequently lead to lens structural protein misfolding and denaturation. Besides, we observed that the mutated CRYBA1 protein would be more aggregate to the submembrane compartment (Fig. 5), most possibly results from 1) the significant decrease in its level in the cell [28] and 2) the structural misfolding of the protein [29]. However, manifestation of the cataract phenotype in a genetically modified animal model, most frequently the mouse model, is needed to provide confirmative evidence of this single residue deletion being the causative mutation of the disease. ...
Background:
Mutations in more than 52 genes have been identified in isolated congenital cataracts, the majority of which are located in crystalline and connexin (gap junction) genes. An in-frame one amino acid deletion in the beta-crystalline gene CRYBA1 has been reported in several different Chinese, Caucasian and Iranian families of congenital cataracts. Further functional studies are needed to confirm the variant pathogenicity.
Methods:
The purpose of this study is to identify the genetic causes that contribute to congenital cataracts with esotropia and nystagmus in a Chinese family. Whole-exome sequencing was performed on samples from all five family members. The two brothers of the father and their daughters were then enrolled in the study, and 40 suspected variants were sequenced among the 9 subjects using Sanger sequencing. The mRNA and protein levels of CRYBA1 in the lens epithelium from cataract patients and normal controls were compared using quantitative polymerase chain reaction (qPCR) and Western blot analyses. The wild-type and mutated forms (p.G91del) of CRYBA1 cDNA were transfected into two types of cell lines, and the expression level of exogenous CRYBA1 was measured by Western blot analysis. The exogenous CRYBA1 proteins were visualized by immunofluorescence staining.
Results:
In this two-generation family, all three descendants inherited congenital cataracts with esotropia and nystagmus from the father, while the mother's lens was normal. After two rounds of sequencing, CRYBA1 (c. 269-271 del, p.G91del) was identified as the mutation responsible for the autosomal dominant congenital cataract in the Chinese family. CRYBA1 showed lower expression in cataract lenses than in control lenses. The deleted form (p.G91del) of CRYBA1 showed lower expression and was more aggregate to the cell membrane than the wild-type CRYBA1.
Conclusions:
We performed molecular experiments to confirm that the p.G91del mutation in CRYBA1 results in abnormal expression and distribution of CRYBA1 protein, and this study could serve as an example of the pathogenicity of an in-frame small deletion in an inherited eye disorder.
... Alzheimer's disease (AD) is the most common cause of dementia in the elderly, the fourth most common cause of death in westem industrialized nations, and one of the major contributors to the global burden of disease (WHO, 2000 (Lovestone & McLoughlin, 2002;Selkoe, 2004). ...
... In neurons, GSK3B is a major Tau kinase, critically involved in the maintenance of the stability of microtubules and, therefore, in the preservation of the structure and function of the neuronal cytoskeleton [28] . Studies have shown that GSK3B is overactive in AD, playing a major role in the hyperphosphorylation of Tau [24,[29][30][31] . In experimental models of AD, GSK3B has been shown to hyperphosphorylate Tau, leading to microtubule disassembly and loss of function [28] . ...
The search for diagnostic and prognostic markers in Alzheimer's disease (AD) has been an area of active research in the last decades. Biochemical markers are correlates of intracerebral changes that can be identified in biological fluids, namely: peripheral blood (total blood, red and white blood cells, platelets, plasma and serum), saliva, urine and cerebrospinal fluid. An important feature of a biomarker is that it can be measured objectively and evaluated as (1) an indicator of disease mechanisms (markers of core pathogenic processes or the expression of downstream effects of these processes), or (2) biochemical responses to pharmacological or therapeutic intervention, which can be indicative of disease modification. Platelets have been used in neuropharmacological models since the mid-fifties, as they share several homeostatic functions with neurons, such as accumulation and release of neurotransmitters, responsiveness to variations in calcium concentration, and expression of membrane-bound compounds. Recent studies have shown that platelets also express several components related to the pathogenesis of AD, in particular to the amyloid cascade and the regulation of oxidative stress: thus they can be used in the search for biomarkers of the disease process. For instance, platelets are the most important source of circulating forms of the amyloid precursor protein and other important proteins such as Tau and glycogen synthase kinase-3B. Moreover, platelets express enzymes involved in membrane homeostasis (e.g., phospholipase A2), and markers of the inflammatory process and oxidative stress. In this review we summarize the available literature and discuss evidence concerning the potential use of platelet markers in AD.
... Throughout the preceding chapters, we have come across numerous examples of disease associated to the presence of large insoluble protein aggregates in cells. The familial, early-onset forms of these 'conformational' disorders have a common denominator: the relevant proteins have mutations that interfere with their folding pathways or inhibit degradation by the proteasome, thereby increasing the tendency of the proteins to aggregate (reviewed in Lovestone and McLoughlin, 2002;Michalik and Van Broeckhoven, 2003;Caughey and Lansbury, 2003). Polyglutamine disorders such as Huntington's disease and a number of spinocerebellar ataxias (SCA) are caused by an expansion of the CAG trinucleotide repeats coding for polyglutamine in the genes that code for the disease protein (huntingtin and ataxins, respectively). ...
Ubiquilin is a ubiquitin-like protein that makes its appearance throughout the
literature in many instances concerning the cellular quality-control system and its
response to unfolded or misfolded proteins. Ubiquilin seems to play a role in the
unfolded protein response, which follows accumulation of aberrant proteins in the ER,
and it is a faithful companion of these proteins along the proteolytic pathways through
which they are eventually disposed of: proteasomal degradation, aggresome
formation, autophagy and delivery to the lysosome. Ubiquilin can accomplish this
complex multi-tasking thanks to its modular structure, which allows it to function as
an adaptor protein in large multi-protein complexes of varying composition and
functionality. Thus, ubiquilin presumably links the misfolded proteins to the
machinery charged with their degradation. The correct and coordinated function of all
the components of the proteolytic system is crucial for cellular homeostasis. When the
system is disrupted or overwhelmed, the accumulation of misfolded and aggregated
proteins can reach toxic levels for the cell. This process is believed to underlie the
pathogenic mechanisms of many neurodegenerative diseases. The accumulation of
degradation-resistant debris in senescent cells, probably due to failure of the qualitycontrol
system, suggests that impaired cellular proteolysis plays a crucial role in
normal aging as well.
... Tau-mediated neurodegeneration can arise from the loss of physiological function and/or the gain-oftoxicity (22,23). Tau aggregation abolishes its MT-stabilizing function and can impair axonal transport (24). ...
The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that anionic lipid membranes can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40s presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40s strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases.
... The toxicity of tau aggregates depends on the loss of physiological functions and the gain of pathological functions (213,382). In the first case, tau aggregates are unable to bind and stabilize MTs, while in the second, soluble oligomeric tau appears to be more toxic than aggregated tau (48). ...
Alzheimer's disease (AD) is the most common cause of dementia and a progressive neurodegeneration that appears to result from multiple pathogenic mechanisms (including protein misfolding/aggregation, involved in both amyloid β-dependent senile plaques and tau-dependent neurofibrillary tangles), metabolic and mitochondrial dysfunction, excitoxicity, calcium handling impairment, glial cell dysfunction, neuroinflammation, and oxidative stress. Oxidative stress, which could be secondary to several of the other pathophysiological mechanisms, appears to be a major determinant of the pathogenesis and progression of AD. The identification of oxidized proteins common for mild cognitive impairment and AD suggests that key oxidation pathways are triggered early and are involved in the initial progression of the neurodegenerative process. Abundant data support that oxidative stress, also considered as a main factor for aging, the major risk factor for AD, can be a common key element capable of articulating the divergent nature of the proposed pathogenic factors. Pathogenic mechanisms influence each other at different levels. Evidence suggests that it will be difficult to define a single-target therapy resulting in the arrest of progression or the improvement of AD deterioration. Since oxidative stress is present from early stages of disease, it appears as one of the main targets to be included in a clinical trial. Exploring the articulation of AD pathogenic mechanisms by oxidative stress will provide clues for better understanding the pathogenesis and progression of this dementing disorder and for the development of effective therapies to treat this disease.
... NFTs are flame-shaped intracytoplasmic inclusions composed of aggregations of hyperphosphorylated tau protein that affect proximal neurites and neuron perikarya and are hallmark pathological features of AD (24). However, tau pathology is also observed in several additional distinct neurodegenerative diseases including parkinson-amyotrophic lateral sclerosis of Guam (GP-ALS) (25), Pick disease (26,27), progressive supranuclear palsy (PSP) (28,29), subacute sclerosing panencephalitis (SSPE) (30,31), frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) (32,33), dementia pugilistica (34), and neurodegeneration with brain iron accumulation type 1 (NBIA) (35), suggesting the possibility of a common underlying pathogenic pathway for these diseases collectively referred to as tauopathies (36). pRb hyperphosphorylated at several sites was found to be elevated and associated with NFTs in AD (22). ...
Retinoblastoma protein (pRb) is a ubiquitous 928-amino acid cell cycle regulatory molecule with diverse biologic activities. One critical function of pRb is the control of the G1-to-S phase checkpoint of the cell cycle. In the hypophosphorylated state, pRb suppresses the activity of E2F transcription factors thereby inhibiting transcription of cell cycle-promoting genes. On phosphorylation, primarily by cyclin-dependent kinases, phosphorylated pRb dissociates from E2F and permits cell cycle progression. We previously found phosphorylated pRb to be intimately associated with hyperphosphorylated tau-containing neurofibrillary tangles of Alzheimer disease (AD), the pathogenesis of which is believed to involve dysregulation of the cell cycle and marked neuronal death. Here, we used immunohistochemistry to investigate the presence of phosphorylated pRb in other distinct neurodegenerative diseases that share the common characteristic of hyperphosphorylated tau pathology and neuronal loss with AD.We found colocalized labeling of tau pathology and phosphorylated pRb in Pick disease and progressive supranuclear palsy (3 cases each), neurodegeneration with brain iron accumulation type 1 (2 cases), and Parkinson-amyotrophic lateral sclerosis of Guam, subacute sclerosing panencephalitis, frontotemporal dementia and Parkinsonism linked to chromosome 17, and dementia pugilistica (1 case each). These observations further implicate aberrant neuronal cell cycle progression in neurodegenerative diseases, particularly tauopathies, and suggest a novel target for therapeutic intervention.
... [1][2][3] Problems related to protein folding, including protein aggregation and misfolding, are involved in diseases such as Alzheimer's, Parkinson's and Huntington's, as well as in prion diseases. [4][5][6][7] Moreover, the efficient production of a large amount of protein is a major focus of biotechnology and postgenome research fields. However, the folding of proteins synthesized in vitro into the correct active structure has proven to be difficult, because partially folded intermediate proteins are considerably hydrophobic and form aggregation and inclusion bodies. ...
Polysaccharide nanogels have been demonstrated to aid the refolding processes of chemically or thermally denatured proteins, a function that is similar to that of natural molecular chaperones. In this study, we examined the possibilities of using the nanogel chaperone system to mediate protein folding in a cell-free (in vitro) protein synthesis system containing transcription/translation factors. High-performance liquid chromatography showed that a polysaccharide nanogel comprising cholesteryl group-bearing pullulan (CHP) trapped unfolded or partially folded green fluorescent protein (GFP) expressed in the cell-free system. The protein release and refolding processes, which are induced by ATP in natural molecular chaperone systems, were also simulated by methyl-β-cyclodextrin (M-β-CD). The CHP nanogels dissociate on complexation with M-β-CD to yield dissociated CHP. Thus, the dissociation of the CHP nanogel–protein complex subsequently allows for the release and folding of GFP. The folding kinetics in the presence of the CHP nanogel and M-β-CD was comparable to that of spontaneous folding in the absence of CHP/M-β-CD, indicating that the CHP nanogels did not affect protein synthesis in the cell-free system, providing correctly folded active proteins.
... The phosphorylation state of Tau is determinant of its ability to stabilize microtubules. In the pathophysiology of AD, hyperactive GSK3B has been associated with the formation of paired helicoidal filaments (PHF-Tau) and, therefore, of neurofibrillary tangles (Frame and Cohen, 2001; Lovestone and McLoughlin, 2002; Kar et al., 2004; Balaraman et al., 2006). In experimental models of AD, GSK3B has been shown to hyperphosphorylate Tau, leading to microtubule disassembly and loss of function (Lovestone et al., 1994). ...
The disruption of glycogen synthase kinase 3-beta (GSK3B) homeostasis has implications in the pathophysiology of neuropsychiatric disorders, namely Alzheimer's disease (AD). GSK3B activity is increased within the AD brain, favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. Such abnormality has also been detected in leukocytes of patients with cognitive disorders. The aim of the present study was to determine the expression of total and phosphorylated GSK3B at protein level in platelets of older adults with varying degrees of cognitive impairment, and to compare GSK3B activity in patients with AD, mild cognitive impairment (MCI) and healthy controls. Sixty-nine older adults were included (24 patients with mild to moderate AD, 22 patients with amnestic MCI and 23 elderly controls). The expression of platelet GSK3B (total- and Ser-9 phosphorylated GSK3B) was determined by Western blot. GSK3B activity was indirectly assessed by means of the proportion between phospho-GSK3B to total GSK3B (GSK3B ratio), the former representing the inactive form of the enzyme. Ser-9 phosphorylated GSK3B was significantly reduced in patients with MCI and AD as compared to controls (p=0.04). Platelet GSK3B ratio was significantly decreased in patients with MCI and AD (p=0.04), and positively correlated with scores on memory tests (r=0.298, p=0.01). In conclusion, we corroborate previous evidence of increased GSK activity in peripheral tissues of patients with MCI and AD, and further propose that platelet GSK may be an alternative peripheral biomarker of this abnormality, provided samples are adequately handled in order to preclude platelet activation.
... The toxicity of Tau aggregates originates the loss of physiological functions and the gain of pathological functions (Lovestone and McLoughlin 2002;Ding and Johnson 2008;Gendron and Petrucelli 2009). In the first case, Tau aggregates are unable to bind and stabilize the MTs, causing an increase of MT instability; this would also increase the expression of Tau protein as a compensatory mechanism. ...
J. Neurochem. (2010) 112, 1353–1367.
We are analyzing the physiological function of Tau protein and its abnormal pathological behavior when this protein is self-assemble into pathological filaments. These aggregates of Tau protein are the main components in many diseases such as Alzheimer’s disease (AD). Recent studies suggest that Tau acquires complex oligomeric conformations which may be toxic. In this review, we emphasized the possible phenomena implicated in the formation of these oligomers. Studies with chemical inductors indicates that the microtubule-binding domain is the most important region involved in Tau aggregation and showed the requirement of a pre-arrange Tau in abnormal conformation to promote self-assembly. Transgenic animal models and AD neuropathology studies showed that post-translational modifications are also implicated in Tau aggregation and neural cell death during AD development. Therefore, we analyzed some events that could be present during Tau aggregation. Finally, we included a brief discussion of the possible relation between glucose metabolism dysfunction in AD, and data of Tau aggregation by using aggregation inhibitors. In conclusion, the process Tau aggregation deserves further investigations to design possible therapeutic targets to inhibit the toxicity of these aggregates and it is possible that could be extended to other diseases with similar etiology.
... Cerebral amyloid diseases are considered to be part of an emerging complex group of chronic and progressive entities collectively known as "Disorders of Protein Folding" that include, among many others, Alzheimer's disease (AD), polyglutamine-repeat disorders, cataracts, amyotrophic lateral sclerosis, Parkinson's disease and other synucleinopathies, tauopathies, a variety of systemic amyloidosis, prion diseases, cerebellar ataxias and type-II diabetes [1][2][3][4]. In all these diseases, through mechanistic pathways poorly understood, soluble proteins normally found in biological fluids change their conformation and form either insoluble aggregates that accumulate intra-or extra-cellularly or fibrillar lesions usually associated with local release of inflammatory mediators, oxidative stress, complement activation, cell toxicity, apoptosis, or a combination thereof resulting in cell damage, organ dysfunction and eventually death. ...
Cerebral amyloid diseases are part of a complex group of chronic and progressive entities bracketed together under the common denomination of protein folding disorders and characterized by the intra- and extracellular accumulation of fibrillar aggregates. Of the more than 25 unrelated proteins known to produce amyloidosis in humans only about a third of them are associated with cerebral deposits translating in cognitive deficits, dementia, stroke, cerebellar and extrapyramidal signs, or a combination thereof. The familial forms reviewed herein, although infrequent, provide unique paradigms to examine the role of amyloid in the mechanism of disease pathogenesis and to dissect the link between vascular and parenchymal amyloid deposition and their differential contribution to neurodegeneration.
... Background Information-Cerebral amyloid diseases are considered part of an emerging complex group of chronic and progressive entities collectively known as "Disorders of Protein Folding" that include sporadic and familial Alzheimer's disease, cerebellar ataxias, Parkinson's and prion diseases, tauopathies, type-II diabetes, systemic amyloidosis, Huntington's disease and cataracts, among others (36)(37)(38)(39). In these diseases, through mechanistic pathways poorly understood, soluble proteins normally found in biological fluids change their conformation and form either insoluble structures that accumulate in the form of either intra-and extra-cellular aggregates or fibrillar lesions usually resulting in cell damage, organ dysfunction and eventually death. ...
Extracellular deposits of amyloid fibrils in the form of parenchymal plaques and cerebrovascular lesions, as well as intracellular accumulation of paired-helical filaments in the form of neurofibrillary tangles (NFT) in selected neuronal populations are the main neuropathologic hallmarks of Alzheimer's disease. Amyloid fibrils composed of polymeric structures of the amyloid-beta (Abeta) concentrate at the center of senile plaques and accumulate in the walls of cerebral blood vessels, exhibiting extensive Congo red/thioflavin S staining. Intraneuronal NFT are composed of building blocks of aberrantly hyperphosphorylated species of the microtubule-associated protein tau, which accumulate in the perinuclear cytoplasm of vulnerable neurons in the form of paired helical filaments (PHF). This unit presents a variety of protocols for the isolation, biochemical analysis, and characterization of amyloid fibrils and neurofibrillary tangles.
... Although composed of unrelated proteins, these aggregates of initially soluble proteins have many common features. They usually have dense fibrillar morphology with a high proportion of b-pleated sheet secondary structure, are often ubiquitinated, and are typically resistant to proteolytic degradation (Lovestone and McLoughlin 2002). Now it is believed, that: (1) a composition of a certain aggregate reflects a whole network of interactions between crucial players in disease pathogenesis; (2) each disease cannot be considered as a single event or gene aberration, rather it is a polygenic disorder or a syndrome; (3) for yet unknown reason, the vast majority of all AD and PD cases are sporadic. ...
At most, many protein-misfolding diseases develop as environmentally induced sporadic disorders. Recent studies indicate that the dynamic interplay between a wide repertoire of noncoding RNAs and the environment play an important role in brain development and pathogenesis of brain disorders. To elucidate this new issue, novel animal models which reproduce the most prominent disease manifestations are required. For this, transgenic Drosophila strains were constructed to express small highly structured, non-coding RNA under control of a heat shock promoter. Expression of the RNA induced formation of intracellular aggregates revealed by Thioflafin T in embryonic cell culture and Congo Red in the brain of transgenic flies. Also, this strongly perturbed the brain control of locomotion monitored by the parameters of sound production and memory retention of young 5-day-old males. This novel model demonstrates that expression of non-coding RNA alone is sufficient to trigger neuropathology.
A person suspected of having Alzheimer’s disease (AD) is clinically diagnosed for the presence of principal biomarkers, especially misfolded amyloid-beta (Aβ) and tau proteins in the brain regions. Existing radiotracer diagnostic tools, such as PET imaging, are expensive and have limited availability for primary patient screening and pre-clinical animal studies. To change the status quo, small-molecular near-infrared (NIR) probes have been rapidly developed, which may serve as an inexpensive, handy imaging tool to comprehend the dynamics of pathogenic progression in AD and assess therapeutic efficacy in vivo. This Perspective summarizes the biochemistry of Aβ and tau proteins and then focuses on structurally diverse NIR probes with coverages of their spectroscopic properties, binding affinity toward Aβ and tau species, and theranostic effectiveness. With the summarized information and perspective discussions, we hope that this paper may serve as a guiding tool for designing novel in vivo imaging fluoroprobes with theranostic capabilities in the future.
The accumulation of ΔK280 tau mutant resulting in neurotoxic oligomeric aggregates is an important but yet mysterious procedure in Alzheimer's disease (AD) development. Recently, we proposed a histidine tautomerization hypothesis of tau fibrillogenesis for the pathobiology of AD and other neuro diseases. However, the influence of neutral histidine tautomeric states on tau mutation is still unclear. Herein, we performed replica-exchange molecular dynamics (REMD) simulations to characterize structural features as well as the mode of toxic action of the ΔK280 tau mutant in the presence of histidine tautomerism. Molecular dynamics (MD) simulation results show that the δε tautomeric isomer (having a distinct global energy minimum) had the highest β-sheet structure, which adopts a sheet-rich conformer and may have significant influence on the structural behaviors of ΔK280 tau monomers. Furthermore, clustering, residual contact map, mobility and structural analysis exhibited that the presence of β-strand interactions between stable lysine 8 (K8)-asparagine 13 (N13) and valine 39 (V39)-tyrosine 43 (Y43) residues plus K31-histidine 32 (H32) and K8-N13 (strand-loop-strand [β-meander] structure) helped δε to form toxic aggregates. Moreover, H299 played a more critical role in the conformational instability of the δε than H268. Overall, the results obtained from this study may be used to arrest neurodegeneration in ΔK280 tau mutation carriers as well as increase the understanding of AD-related tau pathogenesis and strengthen the histidine tautomerism hypothesis of misfolded peptide accumulation.
The central event in the pathogenesis of prion disease is the conversion of PrP, the prion protein, from its normal cellular form (PrPC) to its abnormal conformer (PrPSc). In an experimental scrapie prion infection, the initial inoculum of PrPSc is cleared relatively rapidly, thus a critical aspect of disease progression in vivo is presumably the ability of infected cells, for example in the nervous system, to convert their uninfected neighbours to stable expression of PrPSc. The mechanisms underlying such cell to cell infection are not understood. In the present study, the transfer of scrapie prion infectivity was studied in dissociated cell culture. Genetically marked target cell lines were derived by transfection of tagged PrP molecules into uninfected cells under neomycin selection. In initial experiments, target cells were exposed to subcellular preparations of prion rods and were converted to stable production of PrPSc. In order to study cell-mediated infection, a system was established whereby scrapie-infected mouse SMB cells are able to convert uninfected target cells following coculture of the two populations. The mechanism of intercellular prion transmission in this culture system was evaluated in a series of experiments, and was found to be dependent on direct cell to cell contact. This system for establishing cell-based infection is effective and requires significantly less PrPSc than conversion by an infected brain homogenate. To facilitate the accurate quantitation of conversion at a cellular level, attempts were made to identify a cytochemical marker for PrPSc-expressing cells. This search was pursued by assessing the reactivity of a range of PrP antibodies and reagents including plasminogen. Data obtained from such studies should enable the derivation of immunoreagents able to reliably discriminate between PrPC and PrPSc at the single cell level.
Dark field microscopy directly detects scattering from a sample, and therefore requires no fluorescent labeling for single molecule detection. The technique has been extensively used for spectral characterization of nanoscopic particles, but its sensitivity to variances in molecular density has not been explored. 3D chromosome topology plays important roles in regulating life machinery. Physical characters of DNA (e.g. persistence length) and its interaction with numerous proteins and polyamines determine chromosome and chromatin 3D topology. We herein demonstrate the capacity of dark field microscopy in real-time monitoring of single DNA molecules’ conformation and dynamics. Without fluorescent staining, DNA molecules remaine intact in physical characters like persistence length, net charges and width of the chain. Excluding of photobleaching and photocleavage effect allows long-term imaging of bare DNA chains at high ionic strength, in shear flows and with the presence of condensing and stretching agents. Furthermore, our studies reveal that brightness of single molecules during dark field imaging is determined by DNA conformation, dark when being stretched and bright upon condensation. The sensitivity of dark field microscopy to ununiform distribution in molecular density allows us to study the dynamic activities of transcriptional factor within cell membrane. In contract to fluorescently tagged antibody, which binds to specific signaling pathway, variances in molecular density across nuclear envelop can better represent cellular responses upon stimulation.
This is the second part of a three-part review series reviewing the most important advances in Alzheimer's disease (AD) research since 2010. This review covers the latest research on genetics and epidemiology. Epidemiological and genetic studies are revealing important insights into the etiology of, and factors that contribute to AD, as well as areas of priority for research into mechanisms and interventions. The widespread adoption of genome wide association studies has provided compelling evidence of the genetic complexity of AD with genes associated with such diverse physiological function as immunity and lipid metabolism being implicated in AD pathogenesis.
Objective: To examine the pattern of atrophy modifications in whole-brain and ventricular regions, using data from serial (multi-timepoint) magnetic resonance imaging (MRI) in Alzheimer’s patients, Mild Cognitive Impairment (MCI) and normal aging. To evaluate a published atrophy-measurement technique: Brain Boundary Shift Integral, determining suitable values for parameters which control the BBSI and investigate its stability over time. To test how brain volume tested with BBSI can identify group membership and progress over time and to examine the relationship between these parameters and the cognitive data available.
Methods: The Brain Boundary Shift Integral was computed by the following stages:
Image Pre-processing
Brain extraction and brain segmentation
Registration (alignment) of each pair of MRI scans
BBSI Calibration Using Simulated Data:
Establishing the optimum boundary region (morphological operators)
Establishing the optimum intensity window for ventricular area
Results: The AD patients differed primarily in the rates of brain atrophy and ventricular enlargement from elderly controls and Mild Cognitive Impairment. These results were analyzed according to time variation from baseline to 24 months by controlling for gender and group differences. The overall volumetric loss was correlated with the performance on cognitive scale.
Conclusions: The pattern of brain volume atrophy has progressively decreased over time factor at both level of total and ventricular volume with a significant difference between AD and normal elderly. The correlation between brain atrophy, ventricular enlargement and cognitive scales reflected the correspondence between the measures of cerebral atrophy monitored in time and the global measures of cognitive scale.
Oculopharyngeal Muscular Dystrophy (OPMD) is a muscle disorder with autosomal dominant inheritance, which becomes symptomatic after the age of 40 with slowly progressive bilateral ptosis, dysphagia and sometimes proximal limb weakness. Objetive: to describe the first cases of OPMD confirmed by molecular test in Argentina. Patients and methods: 2 patients from different families with prior diagnostic of polymyositis and myasthenia gravis, for suspect OPMD, we did molecular test. Genomic DNA was extracted from blood lymphocytes according to standard protocols, and amplified by polymerase chain reactions the first exon of the PABPN1 gene. Both cases showed allele 1: 6 repeat and allele 2: 9 repeat. Discussion: to arrive clinical diagnostic of OPMD, is important know the previous basic aspects. The diagnostic gold standart technique is the molecular test that permit us avoid invasive and expensive unnecessary studies, uncorrected drugs use, preclinical diagnosis in unaffected family members and the chance of inclusion in current treatment trails.
Neurodegenerative diseases are characterized by progressive dysfunction and death of cells that frequently affect specific neural systems, thereby implying some form of selective vulnerability. Recent studies have provided insight into the basic processes common to neurodegeneration and into cell-death programs and their roles in the complex group of disorders that are briefly reviewed in this chapter. The major basic processes inducing neurodegeneration are considered multifactorial ones caused by genetic, environmental, and endogenous factors related to aging. Converging lines of investigation have revealed potential common pathogenic mechanisms underlying many diverse neurodegenerative disorders, including abnormal protein dynamics; oxidative stress (OS) and formation of free radicals/reactive oxygen species (ROS); impaired bioenergetics and mitochondrial dysfunctions; fragmentation of neuronal Golgi apparatus (GAs); disruption of cellular/axonal transport; neuroinflammatory/neuro-immune processes; dysfunction of neurotrophins; and actions and mutations of molecular chaperones. These processes and their role in neurodegenerative diseases are briefly discussed in the chapter.
Multiple sclerosisSchilder's disease (diffuse cerebral sclerosis, encephalitis periaxialis diffusa, myelinoclastic diffuse sclerosis)Tuberous sclerosis (tuberose sclerosis, epiloia)NeurofibromatosisFriedreich's ataxiaMotor neurone disease (amyotrophic lateral sclerosis, Lou Gehrig's disease)Myasthenia gravisProgressive muscular dystrophiesMyotonic dystrophiesNeuropsychiatric manifestations of carcinomaNormal-pressure hydrocephalusOther disorders affecting the CNSAcknowledgementsReferences
The amygdala exhibits significant pathological changes in Parkinson’s disease, including atrophy and Lewy body (LB) formation. Amygdala pathology has been suggested to contribute to some clinical features of Parkinson’s disease, including deficits of olfaction and facial expression. The degree of neuronal loss in amygdala subnuclei and the relationship with LB formation in non‐demented Parkinson’s disease cases have not been examined previously. Using stereological methods, the volume of neurones and the number of neurones in amygdala subdivisions were estimated in 18 prospectively studied, non‐demented patients with Parkinson’s disease and 16 age‐ and sex‐matched controls. Careful exclusion (all cortical disease) and inclusion (non‐demented, levodopa‐responsive, idiopathic Parkinson’s disease or controls) criteria were applied. Seven Parkinson’s disease cases experienced well‐formed visual hallucinations many years after disease onset, while nine Parkinson’s disease cases and three controls were treated for depression. Anatomically, the amygdala was subdivided into the lateral nucleus, the basal (basolateral and basomedial) nuclei and the corticomedial (central, medial and cortical nuclei) complex. LB and Lewy neurites were identified by immunohistochemistry for α‐synuclein and ubiquitin and were assessed semiquantitatively. LB were found throughout the amygdala in Parkinson’s disease, being present in ∼4% of neurones. Total amygdala volume was reduced by 20% in Parkinson’s disease ( P = 0.02) and LB concentrated in the cortical and basolateral nuclei. Lewy neurites were present in most cases but did not correlate with any structural or functional variable. Amygdala volume loss was largely due to a 30% reduction in volume ( P = 0.01) and the total estimated number of neurones ( P = 0.007) in the corticomedial complex. The degree of neurone loss and the proportion of LB‐containing neurones in the cortical nucleus within this complex were constant across Parkinson’s disease cases and neither variable was related to disease duration ( R 2 0.5). The cortical nucleus has major olfactory connections and its degeneration is likely to contribute to the early selective anosmia common in Parkinson’s disease. There was a small reduction in neuronal density in the basolateral nucleus in all Parkinson’s disease cases, but no consistent volume or cell loss within this region. However, the proportion of LB‐containing neurones in the basolateral nucleus was nearly doubled in cases that exhibited visual hallucinations, suggesting that neuronal dysfunction in this nucleus contributes to this late clinical feature. Detailed quantitation of the other amygdala subdivisions failed to reveal any other substantial anomalies or any associations with depression. Thus, the impact of Parkinson’s disease on the amygdala is highly selective and correlates with both early and late clinical features.
Substantial genetic, biochemical, and in vivo data indicate that progressive accumulation of amyloid-β (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Historically centered in the importance of parenchymal plaques, the role of cerebral amyloid angiopathy (CAA)-a frequently neglected amyloid deposit present in >80% of AD cases-for the mechanism of disease pathogenesis is now starting to emerge. CAA consistently associates with microvascular modifications, ischemic lesions, micro- and macro-hemorrhages, and dementia, progressively affecting cerebral blood flow, altering blood-brain barrier permeability, interfering with brain clearance mechanisms and triggering a cascade of deleterious pro-inflammatory and metabolic events that compromise the integrity of the neurovascular unit. New evidence highlights the contribution of pre-fibrillar Aβ in the induction of cerebral endothelial cell dysfunction. The recently discovered interaction of oligomeric Aβ species with TRAIL DR4 and DR5 cell surface death receptors mediates the engagement of mitochondrial pathways and sequential activation of multiple caspases, eliciting a cascade of cell death mechanisms while unveiling an opportunity for exploring mechanistic-based therapeutic interventions to preserve the integrity of the neurovascular unit.
The interactions of non‐ionic surfactant Triton X‐100 and the coat protein of tobacco mosaic virus, which is an established model for both ordered and non‐ordered protein aggregation, were studied using turbidimetry, differential scanning calorimetry, isothermal titration calorimetry, and dynamic light scattering. It was found that at the critical aggregation concentration (equal to critical micelle concentration) of 138 × 10 ⁻⁶ M , Triton X‐100 induces partial denaturation of tobacco mosaic virus coat protein molecules followed by protein amorphous aggregation. Protein aggregation has profound ionic strength dependence and proceeds due to hydrophobic sticking of surfactant‐protein complexes (start aggregates) with initial radii of 46 nm. It has been suggested that the anionic surfactant sodium dodecyl sulfate forms mixed micelles with Triton X‐100 and therefore reverses protein amorphous aggregation with release of protein molecules from the amorphous aggregates. A stoichiometric ratio of 5 was found for Triton X‐100‐sodium dodecyl sulfate interactions.
magnified image
Alzheimer's disease is a progressive neurodegenerative disease affecting approximately 8 % of the population over 65 and 30 % of the population over 80 in those countries with a life expectancy at birth as high as ours, with an elevated economic, social and mainly human cost. There is an urgent need to make researches directed to estimate the prevalence and incidence of the disease, to know its risk factors, and to quantify the impact on family and society, which will allow to establish strategies directed to this population and its family. The highest influence on the early diagnosis, the medical attention given to patients and their families and its direct repercussion on the quality of life, will depend on the training and updating of the primary health care team. That's why those aspects connected with the epidemiology and the risk factors, the diagnosis and the current strategies of treatment of this increasing health problem are dealt with in the present review.
Se revisa el concepto de normalidad cognitiva, alteracion cognitiva y el de demencia. Se analizan los principales diagnosticos diferenciales del sindrome de la demencia, la evaluacion de estos pacientes, las principales causas que la determinan, su coste y terapia general.
This chapter presents and discusses different strategies to dissect genetically the molecular mechanisms of Alzheimer’s disease.
First, a short preamble sketches the common phenotypical features of Alzheimer’s disease. Second, a basic introduction defines
the characteristics of a good animal model along with the different molecular strategies commonly used to develop transgenic
animals. Third, an exclusive census of genetically modified mouse models describes some of the remarkable characteristics
observed in these animals and tries to weigh the pros and cons of each of them. Finally, a brief note highlights gene-environment
interactions relevant to the field of genetic mouse models and Alzheimer’s disease. All sections are illustrated with examples
focusing on behavioral phenotypes with a specific interest on learning and memory.
Advances in molecular genetics over the last decade have resulted in the identification of genetic mutations responsible for
several inherited neurological diseases. Not only has cloning of these genes led to methods for diagnosis of patients and
identification of carriers but also to the establishment of animal and cell culture models to study mechanisms by which mutant
proteins induce toxicity in vulnerable cell types. Early neuropathological studies of autopsy tissue from patients with degenerative
neurological diseases commonly revealed the presence of inclusion bodies in affected neuronal populations (see
Table 1; 1–44). These include tangles and plaques in Alzheimer’s disease, Lewy bodies in Parkinson’s disease, and nuclear or cytoplasmic
aggregates in the trinucleotide repeat diseases (spinal bulbar muscular atrophy, Huntington’s disease, spinocerebellar ataxia
1 and 3, dentato-pallidoluysian atrophy) and cytoplasmic inclusions in familial and sporadic motor neuron diseases (45,46). That similar inclusions are observed in both sporadic and hereditary forms of neurological diseases suggested that similar
pathways might be involved in pathogenesis whether protein abnormalities result from inherited sequence differences, DNA damage,
or posttranslational modifications. The presence in inclusions of ubiquitin, a stress protein required for targeting abnormal
proteins for degradation, suggested failure of proteolytic processing to rid cells of aberrant proteins. However, the primary
or secondary role of these inclusions in the pathogenesis of disease could not be surmised from studies of postmortem tissue
at end-stage disease.
Table 1
Examples of Proteotoxicants Resulting in Genetic Mutations Responsible for Human Neurological Disease
Mutant protein
Human disease
Inclusion bodies
Cells most affected
Ref.
Amyloid precursor protein
Alzheimer’s
Extracellular β-amyloid in plagues, neurofibrillary tangles
Limbic and association cortices, hippocampus
1–4
Tau
Frontotemporal dementias Multisystem atrophy
Paired helical filaments in neurofibrillary tangles
Frontotemporal cortical neurons
5–7
Presenilin 1 and 2
Alzheimer’s
Amyloid plaques
Limbic and association cortices, hippocampus
3,4,8–10
α -Synuclein
Parkinson’s Lewy body dementia
Lewy bodies
Substantia nigra Cortical pyramidal neurons
6,11
Cu/Zn-superoxide dismutase (SOD-1)
Chromosome 21-linked amyotrophic lateral sclerosis (ALS)
Cytoplasmic inclusions
Upper and motor motor neurons, astrocytes
12–15
High-molecular-weight neurofilament protein (NF-H)
Rare cases of familial ALS
Hyaline and keinlike inclusions, Bunina bodies
Upper and lower motor neurons
16
*Huntingtin
Huntington’s
Nuclear and cytoplasmic inclusions
Striatum, cerebral cortex
17–20
*Androgen receptor
Kennedy’s disease
Nuclear and cytoplasmic inclusions
Lower motor neurons, dorsal root ganglia
18–22
*Ataxin-1
Spinocerebellar ataxia (SCA1)
Eosinophillic spheroids, nuclear inclusion body
Cerebellar Purkinje, dentate nucleus, brainstem
18–20,23
*Ataxin-2
SCA2
Increased mutant protein, but no inclusions
Cerebellar Purkinje, brain-stem, fronto- temporal lobes
18–20, 24–26
*Ataxin-3
SCA3 / Machado-Joseph disease
Nuclear inclusions
Cerebellar dentate neurons, basal ganglia, brainstem, spinal cord
18–20, 27
8α1A-subunit of voltage-dependent calcum channel
SCA6
Cytoplasmic inclusions
Cerebellar Purkinje and Granule neurons, dentate nucleus, inferior olive
18–20, 28,29
*Ataxin-7
SCA7
Nuclear inclusion
Cerebellum, brainstem, macula, visual cortex
18–20, 30
*Atrophin-1
Dentorubropallidoluysianatrophy
Nuclear inclusion
Cerebellum, cerebral cortex, basal ganglia
18–20, 31,32
* *Poly(A)binding protein 2
Oculopharyngeal dystrophy
Nuclear inclusion
Skeletal muscle
33
Neuroserpin
Familial dementia/progressive myoclonus epilepsy
Collins bodies
Cortical neurons, subcortical nuclei
34–36
Prion protein
Creutzfeld-Jacob (CJD), Gerstmann-Stäussler-Scheinker disease
PrPScdeposition in plaques
Cortex, basal ganglia Cerebellum, cerebrum, brainstem
2,37– 39
Fatal familial insomnia
Kuru
Multiple
Cerebellum, cerebrum, brainstem
PMP22, P0
Charcot-Marie-Tooth
Accumulation in endoplasmic reticulum
Schwann cells
40–44
Note: Trinucleotide repeat diseases with expansion of *polyglutamine or * *polyalanine tracts.
This study demonstrates the power of a novel proteomic approach developed for the detection and identification of biological markers in body fluids. The goal was to observe alterations in the protein patterns of cerebrospinal fluid (CSF) related to amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder with unknown etiology. In the experiments, tryptic digests of CSF from patients and healthy controls were analyzed by on-line capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. (FT-ICR MS) Typically, around 4000 peptides were detected in one such experiment, and a pattern recognition program was constructed for the data analysis to distinguish mass chromatograms from patients and controls. This strategy was evaluated comparing the peptide patterns of CSF spiked in vitro with a biomarker, with control CSF. The patterns were clearly separated and the tryptic peptides of the biomarker were successfully selected as characteristic peaks. Hence, the method was applied to compare mass chromatograms of CSF from 12 ALS-patients and 10 matched healthy controls. While no biomarker alone could be identified from the characteristic peaks, we were able to assign 4 out of 5 unknown samples correctly (i.e., 80% correctly diagnosed, 20% false-negative), and it would be 100% if we reject a possible outlier believed to be caused by an occlusion in the spinal CSF compartment. These findings are very promising, although the clinical relevance is not fully established due to the low number of unknown samples analyzed. In addition to the diagnostic potential, these results may be important steps towards understanding the neurodegenerative process.
The aggregation and accumulation of the microtubule-associated protein (Tau) is a pathological hallmark of Alzheimer disease (AD) and many neurodegenerative diseases. For a long time research has focused on neurofibrillary tangles (NFTs) and other large meta-stable inclusions composed of aggregated hyperphosphorylated tau protein. The correlation between these structures and disease progression produced conflicting results; moreover, the mechanism of their formation remains poorly understood. Lately, the significance and toxicity of NFTs have been challenged and a new aggregated tau entity has emerged as the true pathogenic species in tauopathies and a possible mediator of Aβ toxicity in AD; specifically, aggregates of a size intermediate between monomers and NFTs the so-called tau oligomers. Tremendous efforts have been devoted toward the optimization of a safe vaccine for AD by targeting Aβ peptide; despite the disappointing results, these studies produced a wealth of useful knowledge, which should be considered in developing tau-based immunotherapy. Herein, we discuss the evidence supporting the critical role of tau oligomers in AD, the potential and challenges for targeting them by immunotherapy as a novel approach for AD treatment.
Caspases cleave several cellular proteins to execute cell death by apoptosis. The identification of novel substrates of caspases could provide an important clue for elucidation of new apoptosis signaling pathways. In this study, we tested whether an amyloid precursor protein (APP) binding protein Fe65 is proteolytically degraded in neuronal cell death by apoptosis, using a neuron-like cell line, human neuroblastoma SH-SY5Y cells. When treated with DNA damaging agents, etoposide (ETP) and camptothecin (CPT), SH-SY5Y cells underwent apoptosis in a dose-dependent manner. Interestingly, Fe65 (97 kDa) was cleaved to a 65 kDa product during DNA damage-induced apoptosis. Furthermore, the cleavage of Fe65 was accompanied by activation of caspases-9 and -3. The restriction cleavage of Fe65 was completely suppressed by the treatment with a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethylketone (z-VAD-fmk). These results reveal the restriction cleavage of Fe65 by caspases during DNA damage-induced apoptosis. Since Fe65 has been shown to suppress APP processing to amyloid β (Aβ) production, our findings may provide a new insight into the molecular mechanism by which DNA damage induces Aβ production and subsequent neuronal cell death in Alzheimer's disease (AD).
Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase that phosphorylate protein substrates involved in Alzheimer's disease (AD), such as microtubule-associated protein tau and amyloid precursor protein (APP). GSK-3β consists of two splice variants; the major short form (GSK-3β1) distributes in many organs and the minor long form (GSK-3β2), whose structural difference is the insert of only 13 amino acid residues to the C-terminal side of the catalytic site of GSK-3β1, is present in central nervous system. However, the physiological significances of the two variants are unclear. Here we examined whether the phosphorylation activities of two variants to tau and APP are different in cells. We found that GSK-3β2 has lower phosphorylation activity to tau at AD-relevant epitope (Ser396) than GSK-3β1 in cells, whereas the two variants exhibit equivalent levels of phosphorylation activities to APP. Recombinant GSK-3β2 has also lower phosphorylation activity to tau than GSK-3β1 in vitro, although the phosphorylation activities of the two variants to a synthetic peptide substrate pGS-2 are comparable. Furthermore, the deletion of the C-terminal tail (CT) of GSK-3β2 resulted in considerable reduction of tau phosphorylation activity as compared with GSK-3β1, suggesting that the lower phosphorylation activity of GSK-3β2 to tau is attributed to weak interaction with tau through its unique higher-order structure of CT constructed by the 13 amino acids insertion. Such information may provide a clue for understanding of the physiological significance of the two splice variants of GSK-3β and a new insight into the regulation of tau phosphorylation in central nervous system.
Alzheimer disease treatment has yet to yield a successful therapy that addresses the source of the damage found in brains. Of the varied proposed theories of AD etiology, reactive oxygen species (ROS) generation is cited as a common factor. Efforts to reduce the pathology associated with ROS via antioxidants therefore offer new hope to patients suffering from this devastative disease.
In addition to a large number of clinical descriptions of atypical cases, recent pathological, biochemical and genetic studies challenge the view that amyotrophic lateral sclerosis (ALS) is a disorder restricted to the pyramidal motor system. Relations between ALS, Parkinson disease, fronto-temporal dementia, progressive supranuclear paralysis, and cortico-basal degeneration have now been identified. We propose a review of the topic and discuss the contribution of various clinical and pathological features leading to consider motoneuron diseases as neurodegenerative processes included in a broad spectrum of tauopathies.
The inherent limitations of genetic analysis in humans and other mammals as well as striking conservation of most genes controlling nervous system functioning in flies and mammals made Drosophila an attractive model to investigate various aspects of brain diseases. Since RNA research has made great progress in recent years here we present an overview of studies demonstrating the role of various non-coding RNAs in neurodegeneration and stress response in Drosophila as a model organism. We put special emphasis on the role of non-coding micro RNAs, hsr-omega transcripts, and artificial small highly structured RNAs as triggers of neuropathology including aggregates formation, cognitive abnormalities and other symptoms. Cellular stress is a conspicuous feature of many neurodegenerative diseases and the production of specialized proteins protects the nerve cells against aggregates formation. Therefore, herein we describe some data implicating various classes of non-coding RNAs in stress response in Drosophila. All these findings highlight Drosophila as an important model system to investigate various brain diseases potentially mediated by some non-coding RNAs including polyglutamine diseases, Alzheimer's disease, Huntigton's disease, and many others.
The struggle to identify susceptibility genes for complex disorders has stimulated geneticists to develop new approaches. One approach that has gained considerable interest is to focus on genetically isolated populations rather than on the general population. There remains much controversy and theoretical debate over the feasibility and advantages of such populations, but recent results speak in favor of the feasibility of this approach, and will be reviewed here.
Fe65 is a multimodular adaptor protein expressed mainly in the nervous system. Fe65 binds to the Alzheimer's disease amyloid precursor protein (APP) and the interaction is mediated via a phosphotyrosine binding domain in Fe65 and the carboxy-terminal cytoplasmic domain of APP. Fe65 modulates trafficking and processing of APP, including production of the beta-amyloid peptide that is believed to be central to the pathogenesis of Alzheimer's disease. Fe65 also facilitates translocation of a carboxy-terminal fragment of APP to the nucleus and is required for APP-mediated transcription events. In addition, Fe65 functions in regulation of the actin cytoskeleton and cell movement. Here we report the distribution profile of Fe65 immunoreactivity in adult mouse brain. Fe65 expression was found to be widespread in neurones in adult brain. The areas of highest expression included regions of the hippocampus in which the earliest abnormalities of Alzheimer's disease are detectable. Fe65 was also highly expressed in the cerebellum, thalamus and selected brain stem nuclei. Fe65 was evident in a sub-set of astrocytes within the stratum oriens and radiatum in the hippocampus. Expression of Fe65 was found to be developmentally regulated with levels reducing after embryonic day 15 and increasing again progressively from post-partum day 10 up to adulthood, a developmental pattern that partially parallels that of APP. These data indicate a widespread distribution of Fe65 in neurones throughout mouse brain and also suggest that Fe65 may have functions independent of APP and any potential role in the pathogenesis of Alzheimer's disease.
Alpha-synuclein is a recently discovered protein that was first identified as the major non amyloid component of senile plaques, the cerebral lesion likely responsible for Alzheimer's disease. The role of alpha-synuclein in another brain disease namely Parkinson's disease, has been more deeply documented. It appears that alpha-synuclein fills up the intracytoplasmic inclusions called Lewy bodies that likely contribute to the etiology of Parkinson's disease. Furthermore, rare familial forms of Parkinson's disease have been shown to be linked to autosomal dominant mutations of alpha-synucleins. Is alpha-synuclein a bridge between Alzheimer's and Parkinson's diseases? Could it be seen as a common denominator for these two neurodegenerative diseases? These issues could be better addressed by further delineating the physiological function of alpha-synuclein and, as a corollary, the dysfunction taking place along with the diseases. Here, I will review the recent advances concerning the physiology of alpha-synuclein and will particularly focus on the post-traductional events leading to drastic biophysical transformations. I will describe recent works suggesting that these modifications directly modulate the normal function of alpha-synuclein, likely accounting for the dysfunction associated with Parkinson's disease and perhaps contributing to Alzheimer's pathology.
Huntington's disease (HD) is one of a number of familial polyglutamine (polyQ) repeat diseases. These neurodegenerative disorders are caused by expression of otherwise unrelated proteins that contain an expansion of a polyQ tract, rendering them toxic to specific subsets of vulnerable neurons. These expanded repeats have an inherent propensity to aggregate; insoluble neuronal nuclear and cytoplasmic polyQ aggregates or inclusions are hallmarks of the disorders [1,2]. In HD, inclusions in diseased brains often precede onset of symptoms, and have been proposed to be involved in pathogenicity [3-5]. Various strategies to block the process of aggregation have been developed in an effort to create drugs that decrease neurotoxicity. A discussion of the effect of antibodies, caspase inhibitors, chemical inhibitors, heat-shock proteins, suppressor peptides and transglutaminase inhibitors upon aggregation and disease is presented.
Amyloid plaques and neurofibrillary tangles are the neuropathological hallmarks of Alzheimer's disease (AD), but no conclusive evidence has emerged showing that these hallmarks are the cause and not a product of the disease. Many studies have implicated oxidation and inflammation in the AD process, and there is growing evidence that abnormalities of lipid metabolism also play a role. Using epidemiology to elucidate risk factors and histological changes to suggest possible mechanisms, the hypothesis is advanced that dietary lipids are the principal risk factor for the development of late-onset sporadic AD. The degree of saturation of fatty acids and the position of the first double bond in essential fatty acids are the most critical factors determining the effect of dietary fats on the risk of AD, with unsaturated fats and n-3 double bonds conferring protection and an overabundance of saturated fats or n-6 double bonds increasing the risk. The interaction of dietary lipids and apolipoprotein E isoforms may determine the risk and rate of sustained autoperoxidation within cellular membranes and the efficacy of membrane repair. Interventions involving dietary lipids and lipid metabolism show great promise in slowing or possibly averting the development of AD, including dietary changes, cholesterol-modifying agents and antioxidants.
Huntington's disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAGpolyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2HSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently trans-fecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2HSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2HSDJ in protein folding.
-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two A peptide isoforms, A40 and A42. Here we report the detergent solubilization and partial characterization of -secretase. The activity of solubilized -secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 × 106. Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.
The accumulation of insoluble protein aggregates in intra and perinuclear inclusions is a hallmark of Huntington's disease
(HD) and related glutamine-repeat disorders. A central question is whether protein aggregation plays a direct role in the
pathogenesis of these neurodegenerative diseases. Here we show by using a filter retardation assay that the mAb 1C2, which
specifically recognizes the elongated polyglutamine (polyQ) stretch in huntingtin, and the chemical compounds Congo red, thioflavine
S, chrysamine G, and Direct fast yellow inhibit HD exon 1 protein aggregation in a dose-dependent manner. On the other hand,
potential inhibitors of amyloid-β formation such as thioflavine T, gossypol, melatonin, and rifampicin had little or no inhibitory
effect on huntingtin aggregation in vitro. The results obtained by the filtration assay were confirmed by electron microscopy, SDS/PAGE, and MS. Furthermore, cell
culture studies revealed that the Congo red dye at micromolar concentrations reduced the extent of HD exon 1 aggregation in
transiently transfected COS cells. Together, these findings contribute to a better understanding of the mechanism of huntingtin
fibrillogenesis in vitro and provide the basis for the development of new huntingtin aggregation inhibitors that may be effective in treating HD.
To elucidate the role of the synaptic protein α-synuclein in neurodegenerative disorders, transgenic mice expressing wild-type
human α-synuclein were generated. Neuronal expression of human α-synuclein resulted in progressive accumulation of α-synuclein—and
ubiquitin-immunoreactive inclusions in neurons in the neocortex, hippocampus, and substantia nigra. Ultrastructural analysis
revealed both electron-dense intranuclear deposits and cytoplasmic inclusions. These alterations were associated with loss
of dopaminergic terminals in the basal ganglia and with motor impairments. These results suggest that accumulation of wild-type
α-synuclein may play a causal role in Parkinson's disease and related conditions.
Much evidence indicates that abnormal processing and extracellular deposition
of amyloid-
Parkinson’s disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic
Lewy bodies, the major component of which are filaments consisting of α-synuclein. Two recently identified point mutations
in α-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood.
Here we show that both wild type and mutant α-synuclein form insoluble fibrillar aggregates with antiparallel β-sheet structure
upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag
time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and
only 100 h for the A53T mutant protein. These data suggest that the formation of α-synuclein aggregates could be a critical
step in PD pathogenesis, which is accelerated by the PD-linked mutations.
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanding CAG repeat coding for polyglutamine in the huntingtin protein. Recent data have suggested the possibility that an N-terminal fragment of huntingtin may aggregate in neurons of patients with HD, both in the cytoplasm, forming dystrophic neurites, and in the nucleus, forming intranuclear neuronal inclusion bodies. An animal model of HD using the short N-terminal fragment of huntingtin has also been found to have intranuclear inclusions and this same fragment can aggregate in vitro . We have now developed a cell culture model demonstrating that N-terminal fragments of huntingtin with expanded glutamine repeats aggregate both in the cytoplasm and in the nucleus. Neuroblastoma cells transiently transfected with full-length huntingtin constructs with either a normal or expanded repeat had diffuse cytoplasmic localization of the protein. In contrast, cells transfected with truncated N-terminal fragments showed aggregation only if the glutamine repeat was expanded. The aggregates were often ubiquitinated. The shorter truncated product appeared to form more aggregates in the nucleus. Cells transfected with the expanded repeat construct but not the normal repeat construct showed enhanced toxicity to the apoptosis-inducing agent staurosporine. These data indicate that N-terminal truncated fragments of huntingtin with expanded glutamine repeats can aggregate in cells in culture and that this aggregation can be toxic to cells. This model will be useful for future experiments to test mechanisms of aggregation and toxicity and potentially for testing experimental therapeutic interventions.
Alpha-2-macroglobulin (alpha-2M; encoded by the gene A2M) is a serum pan-protease inhibitor that has been implicated in Alzheimer disease (AD) based on its ability to mediate the clearance and degradation of A beta, the major component of beta-amyloid deposits. Analysis of a deletion in the A2M gene at the 5' splice site of 'exon II' of the bait region (exon 18) revealed that inheritance of the deletion (A2M-2) confers increased risk for AD (Mantel-Haenzel odds ratio=3.56, P=0.001). The sibship disequilibrium test (SDT) also revealed a significant association between A2M and AD (P=0.00009). These values were comparable to those obtained for the APOE-epsilon4 allele in the same sample, but in contrast to APOE-epsilon4, A2M-2 did not affect age of onset. The observed association of A2M with AD did not appear to account for the previously published linkage of AD to chromosome 12, which we were unable to confirm in this sample. A2M, LRP1 (encoding the alpha-2M receptor) and the genes for two other LRP ligands, APOE and APP (encoding the amyloid beta-protein precursor), have now all been genetically linked to AD, suggesting that these proteins may participate in a common neuropathogenic pathway leading to AD.
We have genotyped 292 affected sibling pairs (ASPs) with Alzheimer's disease (AD) according to NINCDS-ADRDA diagnostic criteria and with onset ages of >/=65 years using 237 microsatellite markers separated by an average distance of 16.3 cM. Data were analysed by SPLINK and MAPMAKER/SIBS on the whole sample of 292 ASPs and subsets of 162 ASPs where both members possessed an apolipoprotein E (APOE)straightepsilon4 allele and 63 pairs where neither possessed anstraightepsilon4 allele. Sixteen peaks with a multipoint lod score (MLS) >1 either in the whole sample, the straightepsilon4-positive or -negative subgroups were observed on chromosomes 1 (two peaks), 2, 5, 6, 9 (two peaks), 10 (two peaks), 12, 13, 14, 19, 21 and X (two peaks). Simulation studies revealed that these findings exceeded those expected by chance, although many are likely to be false positives. The highest lod scores on chromosomes 1 (MLS 2.67), 9 (MLS 2.38), 10 (MLS 2.27) and 19 (MLS 1.79) fulfilLander and Kruglyak's definition of 'suggestive' in that they would be expected to occur by chance once or less per genome scan. Several other peaks were only marginally less significant than this, in particular those on chromosomes 14 (MLS 2.16), 5 (MLS 2.00), 12, close to alpha2-macroglobulin (MLS 1.91), and 21, close to amyloid precursor protein (MLS 1.77). This is the largest genome scan to date in AD and shows for the first time that this is a genetically complex disorder involving several, perhaps many, genes in addition to APOE. Moreover, our data will be of interest to those hoping to identify positional candidate genes using information emerging from neurobiological studies of AD.
Huntington's disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAG/polyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2/HSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2/HSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2/HSDJ in protein folding.
The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death.
The prion protein (PrP) is central to the aetiology of the prion diseases, transmissible neurodegenerative conditions of humans and animals. PrP null mice show abnormalities of synaptic neurophysiology, in particular weakened GABAA receptor-mediated fast inhibition and impaired long-term potentiation in the hippocampus. Here we demonstrate that this PrP null phenotype is rescued in mice with a high copy number of a transgene encoding human PrP but not in low copy number mice, confirming the specificity of the phenotype for loss of function of PrP. The ability of human PrP to compensate for loss of murine PrP will allow direct study of the functional consequences of the 18 human PrP mutations, which cause the inherited prion diseases; this phenotype can now form the basis of the first functional assay for PrP.
Members of the Notch family of transmembrane receptors mediate a number of developmental decisions in invertebrates. In order to study Notch function in a vertebrate organism, we have mutated the Notch1 gene of the mouse. Notch1 gene function is required for embryonic survival in the second half of gestation. In the first half of gestation, we have found no effect of the mutation on the normal programs of neurogenesis, myogenesis or apoptosis. We conclude that Notch1 function is not essential for these processes, at least in early postimplantation development. However, we have found that somitogenesis is delayed and disorganized in Notch1 mutant embryos. We propose that Notch1 normally coordinates the process of somitogenesis, and we provide a model of how this might occur.
The apolipoprotein E (apoE) type 4 allele (APOE4) is a susceptibility gene for late-onset familial and sporadic Alzheimer disease. ApoE is found in some neurofibrillary tangle-bearing neurons, one of the major pathologic hallmarks of the disease. Neurofibrillary tangles contain paired helical filaments formed from hyperphosphorylated microtubule-associated protein tau. In vitro, tau binds avidly to apoE3, but not to apoE4, forming a bimolecular complex. Tau phosphorylated with a brain extract does not bind either isoform. ApoE3 binds to the microtubule-binding repeat region of tau, which is also the region that is thought to cause self-assembly into the paired helical filament. Binding studies with fragments of ApoE demonstrate that the tau-binding region of apoE3 corresponds to its receptor-binding domain and is distinct from the region that binds lipoprotein particles or beta/A4 peptide. Isoform-specific interactions of apoE with tau may regulate intraneuronal tau metabolism in Alzheimer disease and alter the rate of formation of paired helical filaments and neurofibrillary tangles.
Apolipoprotein E (apoE), a plasma apolipoprotein that plays a central role in lipoprotein metabolism, is localized in the senile plaques, congophilic angiopathy, and neurofibrillary tangles of Alzheimer disease. Late-onset familial and sporadic Alzheimer disease patients have an increased frequency of one of the three common apoE alleles, epsilon 4, suggesting apoE4 is associated with increased susceptibility to disease. To follow up on this suggestion, we compared the binding of synthetic amyloid beta (beta/A4) peptide to purified apoE4 and apoE3, the most common isoform. Both isoforms bound synthetic beta/A4 peptide, the primary constituent of the plaque and angiopathy, forming a complex that resisted dissociation by boiling in SDS. Oxygen-mediated complex formation was implicated because binding was increased in oxygenated buffer, reduced in nitrogen-purged buffer, and prevented by reduction with dithiothreitol or 2-mercaptoethanol. Binding of beta/A4 peptide was saturable at 10(-4) M peptide and required residues 12-28. Examination of apoE fragments revealed that residues 244-272 are critical for complex formation. Both oxidized apoE4 and apoE3 bound beta/A4 peptide; however, binding to apoE4 was observed in minutes, whereas binding to apoE3 required hours. In addition, apoE4 did not bind beta/A4 peptide at pH < 6.6, whereas apoE3 bound beta/A4 peptide from pH 7.6 to 4.6. Together these results indicate differences in the two isoforms in complexing with the beta/A4 peptide. Binding of beta/A4 peptide by oxidized apoE may determine the sequestration or targeting of either apoE or beta/A4 peptide, and isoform-specific differences in apoE binding or oxidation may be involved in the pathogenesis of the intra- and extracellular lesions of Alzheimer disease.
Amyloid beta-peptide (A beta) deposition in senile plaques and cerebral vessels is a neuropathological feature of Alzheimer disease (AD). We examined the possibility that commonly observed variability in A beta deposition in late-onset AD might be related to apolipoprotein E genotype (APOE gene; the two most common alleles are 3 and 4), since APOE4 is a susceptibility gene for late-onset AD and apolipoprotein E interacts strongly with A beta in vitro. In an autopsy series of brains of late-onset AD patients, we found a strong association of APOE4 allele with increased vascular and plaque A beta deposits. Late-onset AD patients with one or two APOE4 alleles have a distinct neuropathological phenotype compared with patients homozygous for APOE3.
Abnormal and excessive accumulation of the amyloid beta-peptide (A beta) in the brain is a major and common characteristic of all Alzheimer's disease (AD) forms irrespective of their genetic background. Insoluble aggregates of A beta are identified as amyloid plaques. These deposits are thought to form when the amount of A beta is increased in the brain parenchyma as a result of either overexpression or altered processing of the amyloid precursor protein (APP). Soluble A beta ending at carboxyl-terminal residue 40 (A beta 40) and, in lesser amount, the form ending at residue 42 (A beta 42), are normal products of the APP metabolism in cell cultures. Increased secretion of soluble A beta 42 has been observed in cells transfected with constructs modeling APP gene mutations of familial forms of AD (refs 4, 5). On the basis of these in vitro data it has been hypothesized that the presence of soluble A beta 42 plays a role in the formation of amyloid plaques. Subjects affected by Down's syndrome (DS) have an increased APP gene dosage and overexpress APP. Apparently because of this overexpression, they almost invariably develop amyloid deposits after the age of 30 years, although they are free of them at earlier ages. Moreover, it has been observed that A beta 42 precedes A beta 40 in the course of amyloid deposition in DS brain. Thus, DS subjects provide the opportunity to investigate in the human brain the metabolic conditions that precede the formation of the amyloid deposits. Here we report that soluble A beta 42 is present in the brains of DS-affected subjects aged from 21 gestational weeks to 61 years but it is undetectable in age-matched controls. It is argued that overexpression of APP leads specifically to A beta 42 increase and that the presence of the soluble A beta 42 is causally related to plaque formation in DS and, likely, in AD brains.
Exonic and intronic mutations in Tau cause neurodegenerative syndromes characterized by frontotemporal de-mentia and filamentous tau protein deposits. Here we describe a K257T missense mutation in exon 9 of Tau. The proband, a 47-yr-old male, presented with severe personality changes followed by semantic memory loss. A diagnosis of Pick's disease was made. The symptoms progressed until death at age 51. The proband's brain showed a marked frontotemporal atrophy that was most pronounced in the temporal lobes. Numerous tau-immunoreactive Pick bodies were present in the neocortex and the hippocampal formation, as well as in some subcortical brain regions. Their appearance and staining characteristics were indistinguishable from those of sporadic Pick's disease. Diffuse staining for hyperphosphorylated tau was also observed in some nerve cell bodies. Immunoblot analysis of sarkosyl-insoluble tau showed 2 major bands of 60 and 64 kDa and 2 very minor bands of 68 and 72 kDa. Upon dephosphorylation, these bands resolved into 6 bands consisting of 3-repeat and 4-repeat tau isoforms, with an overall preponderance of 3-repeat tau. Isolated tau filaments were narrow, irregularly twisted ribbons. Biochemically, recombinant tau proteins with the K257T mutation showed a reduced ability to promote microtubule assembly, suggesting that this may be the primary effect of the mutation. In addition, the K257T mutation was found to stimulate heparin-induced assembly of 3-repeat tau into filaments. Taken together, the present findings indicate that the K257T mutation in Tau can cause a dementing condition similar to Pick's disease.
A monoclonal antibody to the microtubule-associated protein tau (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to tau, electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to tau-labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that tau in Alzheimer brain is an abnormally phosphorylated protein component of PHF.
Eighty-three brains obtained at autopsy from nondemented and demented individuals were examined for extracellular amyloid deposits and intraneuronal neurofibrillary changes. The distribution pattern and packing density of amyloid deposits turned out to be of limited significance for differentiation of neuropathological stages. Neurofibrillary changes occurred in the form of neuritic plaques, neurofibrillary tangles and neuropil threads. The distribution of neuritic plaques varied widely not only within architectonic units but also from one individual to another. Neurofibrillary tangles and neuropil threads, in contrast, exhibited a characteristic distribution pattern permitting the differentiation of six stages. The first two stages were characterized by an either mild or severe alteration of the transentorhinal layer Pre-alpha (transentorhinal stages I-II). The two forms of limbic stages (stages III-IV) were marked by a conspicuous affection of layer Pre-alpha in both transentorhinal region and proper entorhinal cortex. In addition, there was mild involvement of the first Ammon's horn sector. The hallmark of the two isocortical stages (stages V-VI) was the destruction of virtually all isocortical association areas. The investigation showed that recognition of the six stages required qualitative evaluation of only a few key preparations.
To learn about the carboxy-terminal extent of amyloid β-protein (Aβ) composition of senile plaques (SPs) in the brain affected with Alzheimer's disease (AD), we employed two end-specific monoclonal antibodies as immunocytochemical probes: one is specific for Aβ40, the carboxyl terminus of Aβ1–40, while the other is specific for Aβ42(43). In the AD cortex, all SPs that were labeled with an authentic antibody were Aβ42(43) positive, while only one-third of which, on the average, were Aβ40 positive. There was a strong correlation between Aβ40 positivity and mature plaques. Two familial AD cortices with the mutation of β-amyloid protein precursor 717 (βAPP717) (Val to lie) showed a remarkable predominance of Aβ42(43)-positive, Aβ40-negative plaques. Diffuse plaques, representing the earliest stage of Aβ deposition, were exclusively positive for Aβ42(43) but completely negative for Aβ40.
Adult human nerve cells contain tau protein, a phosphorylated microtubule-associated protein, that is hyperphosphorylated in the fetus and in patients with Alzheimer's disease. Hyperphosphorylation, which diminishes the microtubule-binding capacity of tau, destabilizes microtubules and may enhance the formation of paired helical filaments that constitute neurofibrillary tangles in Alzheimer's disease. Here, we use phosphorylation-dependent anti-tau antibodies to detect specific epitopes that characterize hyperphosphorylated tau. Our demonstration of intracellular tangles containing full-length tau that are not immunolabeled by these antibodies suggests that hyperphosphorylation of tau is not obligatory in the formation of neurofibrillary tangles in Alzheimer's disease.
A key step in the activation of heterodimeric integrin adhesion receptors is the transmission of an agonist-induced cellular signal from the short - and/or -cytoplasmic tails to the extracellular domains of the receptor. The structural details of how the cytoplasmic tails mediate such an inside-out signaling process remain unclear. We report herein the NMR structures of a membrane-anchored cytoplasmic tail of the IIb-subunit and of a mutant IIb-cytoplasmic tail that renders platelet integrin alphaIIbbeta3 constitutively active. The structure of the wild-type alphaIIb-cytoplasmic tail reveals a "closed" conformation where the highly conserved N-terminal membrane-proximal region forms an alpha-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix. The structure of the active mutant is significantly different, having an "open" conformation where the interactions between the N-terminal helix and C-terminal region are abolished. Consistent with these structural differences, the two peptides differ in function: the wild-type peptide suppressed alphaIIbbeta3 activation, whereas the mutant peptide did not. These results provide an atomic explanation for extensive biochemical/mutational data and support a conformation-based "on/off switch" model for integrin activation.
Abundant deposition of amyloid-β (in senile plaques) is a key neuropathological hallmark of Alzheimer's disease (AD). The major component of plaques is the 39– to 43–amino acid amyloid-β peptide. Amyloid-β is generated via proteolytic processing of the amyloid precursor protein (APP) by two
Mutations in the presenilin-1 (PS1) gene are associated with
Alzheimer's disease and cause increased secretion of the neurotoxic
amyloid- peptide (A). Critical intramembraneous aspartates
at residues 257 and 385 are required for the function of PS1 protein. Here
we investigate the biological function of a naturally occurring PS1 splice
variant (PS1 exon 8), which lacks the critical aspartate 257. Cell
lines that stably express PS1 exon 8 or a PS1 protein in which aspartate
residue 257 is mutated secrete significant levels of A, whereas A
generation is severely reduced in cells transfected with PS1 containing a
mutation of aspartate 385. In contrast, endoproteolytic processing of Notch
is almost completely inhibited in cell lines expressing any of the PS1 variants
that lack one of the critical aspartates. These data indicate that PS1 may
differentially facilitate -secretase-mediated generation of A and
endoproteolysis of Notch.
Lewy bodies, a defining pathological characteristic of Parkinson's disease and dementia with Lewy bodies (DLB)1, 2, 3, 4, constitute the second most common nerve cell pathology, after the neurofibrillary lesions of Alzheimer's disease. Their formation may cause neurodegeneration, but their biochemical composition is unknown. Neurofilaments and ubiquitin are present5, 6, 7, 8, but it is unclear whether they are major components of the filamentous material of the Lewy body9,10. Here we describe strong staining of Lewy bodies from idiopathic Parkinson's disease with antibodies for -synuclein, a presynaptic protein of unknown function which is mutated in some familial cases of the disease11. -Synuclein may be the main component of the Lewy body in Parkinson's disease. We also show staining for -synuclein of Lewy bodies from DLB, indicating that the Lewy bodies from these two diseases may have identical compositions.
Constitutive amyloid precursor protein (APP) metabolism results in the generation of soluble APP (APPs) and Aβ peptides, including
Aβ40 and Aβ42–the major component of amyloid plaques in Alzheimer’s disease brain. The phosphotyrosine binding (PTB) domain
of X11 binds to a peptide containing a YENPTY motif found in the carboxyl terminus of APP. We have cloned the full-lengthX11 gene now referred to as X11α.Coexpression of X11α with APP results in comparatively greater levels of cellular APP and less APPs, Aβ40, and Aβ42 recovered
in conditioned medium of transiently transfected HEK 293 cells. These effects are impaired by a single missense mutation of
either APP (Y682G within the YENPTY motif) or X11α (F608V within the PTB domain), which diminishes their interaction, thus
demonstrating specificity. The inhibitory effect of X11α on Aβ40 and Aβ42 secretion is amplified by coexpression with the
Swedish mutation of APP (K595N/M596L), which promotes its amyloidogenic processing. Pulse-chase analysis demonstrates that
X11α prolongs the half-life of APP from ∼2 h to ∼4 h. The effects of X11α on cellular APP and APPs recovery were confirmed
in a 293 cell line stably transfected with APP. The specific binding of the PTB domain of X11α to the YENPTY motif-containing
peptide of APP appears to slow cellular APP processing and thus reduces recovery of its soluble fragments APPs, Aβ40, and
Aβ42 in conditioned medium of transfected HEK 293 cells. X11α may be involved in APP trafficking and metabolism in neurons
and thus may be implicated in amyloidogenesis in normal aging and Alzheimer’s disease brain.
Exonic and intronic mutations in Tou cause familial neurodegenerative syndromes characterized by frontotemporal dementia and dysfunction of multiple cortical and subcortical circuits. Here we describe a G389R mutation in exon 13 of Tau. When 38 years old, the proband presented with progressive aphasia and memory disturbance, followed by apathy, indifference, and hyperphagia. Repeated magnetic resonance imaging showed the dramatic progression of cerebral atrophy. Positron emission tomography revealed marked glucose hypometabolism that was most severe in left frontal, temporal, and parietal cortical regions. Rigidity, pyramidal signs and profound dementia progressed until death at 43 years of age. A paternal uncle, who had died at 43 years of age, had presented with similar symptoms. The proband's brain showed numerous tauimmunoreactive Pick body-like inclusions in the neocortex and the fascia dentata of the hippocampus. In addition, large numbers of tau-positive filamentous inclusions were present in axons in the frontal, temporal, and parietal lobes. Immunoblot analysis of sarkosyl-insoluble tau showed 2 major bands of 60 and 64 kDa. Upon dephosphorylation, these bands resolved into 4 bands consisting of three- and four-repeat tau isoforms. Most isolated tau filaments were straight and resembled filaments found in Alzheimer disease and some frontotemporal dementias with tau mutations. A smaller number of twisted filaments was also observed. Biochemically, recombinant tau proteins with the G389R mutation showed a reduced ability to promote microtubule assembly, suggesting that this may be the primary effect of the mutation. Taken together, the present findings indicate that the G389R mutation in Tou can cause a dementing condition that closely resembles Pick's disease.
(C) 1999 American Association of Neuropathologists, Inc
Abstract : The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective α-secretase-derived secreted APP (APPsα) and the Alzheimer’s disease-associated β-amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the C-terminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin-coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a threefold increase in the amount of APPsα recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPsα secreted is due neither to a simple increase in the steady-state levels of APP nor to activation of the protein kinase C-regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway.
Abundant tau-positive neurofibrillary lesions constitute a defining neuropathological characteristic of Alzheimer's disease. Filamentous tau pathology is also central to a number of other dementing disorders, such as Pick's disease, progressive supranuclear palsy, corticobasal degeneration and familial frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). The discovery of mutations in the tau gene in FTDP-17 has firmly established the relevance of tau pathology for the neurodegenerative process. Experimental studies have provided a system for the assembly of full-length tau into Alzheimer-like filaments, providing an assay for the testing of compounds that inhabit the formation of tau filaments.Trends Neurosci. (1998) 21, 428–433
A genetic classification of Alzheimer disease(s) (AD) is presented. We describe a potential metabolic process in individuals who inherit apolipoprotein E-ϵ4 (APOE4, gene; apoE4, protein) alleles, leading to increased risk and earlier age of onset of late-onset Alzheimer disease. Apolipoprotein E-ϵ3 (apoE3) binds to tau protein, possibly slowing the initial rate of tau phosphorylation and self-assembly into paired helical filments (PHFs); apoE4 does not bind tau. Tau promotes microtubule assembly and stabilizes microtubules; hyperphosphorylated tau does not bind, thereby destabilizing microtubules. Hyperphosphorylated tau may self-assemble into PHFs. Over time a bias toward destabilization of microtubules and the formation of neurofibrillary tangles may occur in individuals who inherit APOE4 alleles, leading to a shorter functional neuronal life span. This hypothesis focuses attention on two important aspects of AD research design: (1) Although the inheritance of APOE4 is associated with increased risk and decreased age of onset, apoE4 does not directly cause the disease. Our data point to the absence of an important function of apoE3 or apoE2 in individuals who do not inherit these alleles as the genetically relevant metabolic factor. This has important implications for design of experiments directed toward understanding the relevant neuronal metabolism. (2) Should this hypothesis be proven and confirmed, targets for pharmaceutical therapy designed to mimic the metabolic function of apoE3 or apoE2 become a realistic preventive strategy.
The importance of apolipoproteins in the central nervous system became increasingly clear with the association in 1993 of the ϵ4 allele of apolipoprotein E with familial and sporadic late-onset Alzheimer's disease. Apolipoprotein E is a ligand for several receptors, most of which are found to some extent in the brain. This review summarizes the various apolipoproteins and lipoprotein receptors found in the brain. A growing body of evidence now implicates irregular lipoprotein metabolism in several neurodegenerative disorders. We then focus on research linking apolipoprotein E and Alzheimer's disease, from clinical studies to biochemical models, which may explain some of the complex neurobiology of this disorder.
APR 1995, RESCUE OF NEUROPHYSIOLOGICAL PHENOTYPE SEEN IN PRP NULL MICE BY TRANSGENE ENCODING HUMAN PRION PROTEIN, (VOL 9, PG 197, 1995)
The most common cause of dementia occurring in mid- to late-life is Alzheimer's disease (AD). Some cases of AD, particularly those of early onset, are familial and inherited as autosomal dominant disorders linked to the presence of mutant genes that encode the amyloid precursor protein (APP) or the presenilins (PS1 or PS2). These mutant gene products cause dysfunction/death of vulnerable populations of nerve cells important in memory, higher cognitive processes, and behavior. AD affects 7-10% of individuals > 65 years of age and perhaps 40% of individuals > 80 years of age. For the late-onset cases, the principal risk factors are age and apolipoprotein (apoE) allele type, with apoE4 allele being a susceptibility factor. In this review, we briefly discuss the clinical syndrome of AD and the neurobiology/neuropathology of the disease and then focus attention on mutant genes linked to autosomal dominant familial AD (FAD), the biology of the proteins encoded by these genes, and the recent exciting progress in investigations of genetically engineered animal models that express these mutant genes and develop some features of AD.
Submitted to the Department of Biological Sciences. Copyright by the author. Thesis (Ph. D.)--Stanford University, 2003.
Eighty-three brains obtained at autopsy from nondemented and demented individuals were examined for extracellular amyloid deposits and intraneuronal neurofibrillary changes. The distribution pattern and packing density of amyloid deposits turned out to be of limited significance for differentiation of neuropathological stages. Neurofibrillary changes occurred in the form of neuritic plaques, neurofibrillary tangles and neuropil threads. The distribution of neuritic plaques varied widely not only within architectonic units but also from one individual to another. Neurofibrillary tangles and neuropil threads, in contrast, exhibited a characteristic distribution pattern permitting the differentiation of six stages. The first two stages were characterized by an either mild or severe alteration of the transentorhinal layer Pre-alpha (transentorhinal stages I-II). The two forms of limbic stages (stages III-IV) were marked by a conspicuous affection of layer Pre-alpha in both transentorhinal region and proper entorhinal cortex. In addition, there was mild involvement of the first Ammon's horn sector. The hallmark of the two isocortical stages (stages V-VI) was the destruction of virtually all isocortical association areas. The investigation showed that recognition of the six stages required qualitative evaluation of only a few key preparations.
The form and distribution of senile plaques (SP) and neurofibrillary tangles (NFT) has been examined in the brains of 13 patients with Down's syndrome (DS), aged less than or equal to 50 years, using immunocytochemical and silver staining procedures. SP become present within the brain before NFT and these appear firstly as fine, even, diffuse areas of anti-amyloid (A4) protein immunoreactivity in the absence of any discernable neuritic change. Later the amount of anti-A4 protein in SP increases and SP show a marked surrounding neuritic response which is detectable using either silver or anti-paired helical filament (PHF) staining methods. At this stage NFT also become detectable within the perikaryon of nerve cells in both the cortex and the subcortex, with the large stellate neurones of layer II of the entorhinal cortex showing an early involvement. By the age of 50 years, most patients are well on the way towards achieving (and some have already achieved) that pattern of SP and NFT morphology and distribution that is typically seen in patients over 50 years of age with DS and in other patients in the general population with Alzheimer's disease.
A monoclonal antibody to the microtubule-associated protein tau (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to tau, electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to tau-labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that tau in Alzheimer brain is an abnormally phosphorylated protein component of PHF.
The protein beta-amyloid is said to be central to the disease process of Alzheimer's disease (AD). Several groups have developed transgenic models that overexpress the amyloid precursor protein or beta-amyloid and then develop AD-like neuropathology. Another report suggests that beta-amyloid accumulation in old dogs correlates with cognitive impairment. However, many other researchers argue that beta-amyloid deposition in senile plaques is a secondary event because plaque numbers in man do not correlate well with cognition. We set out to analyse this conumdrum in man. We selected 16 mild to severely demented AD cases on the basis of mini-mental state exam scores (MMSE; n = 16). We also included 4 controls who represented the upper range of cognitive ability. We used a computer-based image analysis of cross-sectional area of the brain occupied by beta-amyloid immunopositive deposition. We used this technique in preference to conventional methods of manual plaque counts and found a strong relation between beta-amyloid load in entorhinal cortex and cognition measured on various scales (r = -0.93 versus the Blessed IMC). Our study suggests that the size of cortical area affected by beta-amyloid deposition is an important factor in the clinical manifestation of dementia, and lends support to the possibility that beta-amyloid is central to the aetiology of AD.
In Alzheimer's disease, synuclein/NAC (non-amyloid beta component of Alzheimer's disease amyloid) proteins are found in presynaptic cholinergic nerve terminals that degenerate early in Alzheimer's disease, and they are also found closely linked to beta-amyloid fibrils in senile plaques. Synuclein/NAC proteins provide a potential molecular link between the degeneration of cholinergic nerve terminals, and the formation of plaques, and might have a primary role in their development.
Specific immunocytochemical methods (AT8) permit evaluation of neuronal changes well before the actual formation of neurofibrillary tangles and neuropil threads. Initial changes are found in the transentorhinal region (temporal lobe). From here the destructive process encroaches upon the entorhinal region, Ammon's horn, and neocortex. Initial changes occur in comparatively young individuals and can also be observed at the same predilection sites in a few species of old aged domestic animals. In a later state of destruction, AT8 immunoreactive neurons develop typical argyrophilic neurofibrillary tangles and neuropil threads. Six stages of disease propagation can be distinguished with respect to the location of the tangle-bearing neurons and the severity of changes (transentorhinal stages I-II: clinically silent cases; limbic stages III-IV: incipient Alzheimer's disease; neocortical stages V-VI: fully developed Alzheimer's disease). Whole mount techniques reveal the lesional pattern of the particularly severely involved superficial entorhinal layer as seen from the free surface of the parahippocampal gyrus. This approach facilitates recognition of even subtle pathologic changes throughout the entire extent of cortical territories such as the transentorhinal and entorhinal regions.
To learn about the carboxy-terminal extent of amyloid beta-protein (A beta) composition of senile plaques (SPs) in the brain affected with Alzheimer's disease (AD), we employed two end-specific monoclonal antibodies as immunocytochemical probes: one is specific for A beta 40, the carboxyl terminus of A beta 1-40, while the other is specific for A beta 42(43). In the AD cortex, all SPs that were labeled with an authentic antibody were A beta 42(43) positive, while only one-third of which, on the average, were A beta 40 positive. There was a strong correlation between A beta 40 positivity and mature plaques. Two familial AD cortices with the mutation of beta-amyloid protein precursor 717 (beta APP717) (Val to Ile) showed a remarkable predominance of A beta 42(43)-positive, A beta 40-negative plaques. Diffuse plaques, representing the earliest stage of A beta deposition, were exclusively positive for A beta 42(43), but completely negative for A beta 40.
The prion protein PrPc is a glycoprotein of unknown function normally found in neurons and glia. It is involved in diseases such as bovine spongiform encephalopathy (BSE), scrapie and Creutzfeldt-Jakob disease. PrPSc, an altered isoform of PrPC that is associated with disease, shows greater protease resistance and is part of the infectious agent, the prion. Prion diseases are characterized by neuronal degeneration, gliosis and accumulation of PrPSc. Mice devoid of PrPC are resistant to scrapie. A fragment of human PrP consisting of amino acids 106-126 that forms fibrils in vitro is toxic to cultured neurons. Here we show that this toxic effect requires the presence of microglia which respond to PrP106-126 by increasing their oxygen radical production. The combined direct and microglia-mediated effects of PrP106-126 are toxic to normal neurons but are insufficient to destroy neurons from mice not expressing PrPC.
Alzheimer's disease (AD) is a heterogeneous dementing disorder of the elderly that is characterized by progressive cognitive impairments and the accumulation of abundant amyloid or senile plaques (SPs) and neurofibrillary tangles (NFTs) as well as the massive loss of neurons in the AD brain. Indeed, a secure diagnosis of AD in patients with a chronic progressive dementia requires evidence of numerous SPs and NFTs in the postmortem brain. Although the deposition of fibrillar amyloid or A beta-peptides in extracellular plaques and the accumulation of tau-rich intraneuronal NFTs are not restricted exclusively to AD, there is a close correlation between the burden of tau-rich neurofibrillary lesions in selected telencephalic regions of the brain and the dementia in AD. Since the formation of neurofibrillary lesions from hyperphosphorylated tau proteins may compromise the function and viability of neurons in the AD brain, this review summarizes recent insights into mechanisms that regulate the phosphorylation state of tau in AD.
Tau protein, which is incorporated into the core of paired helical filaments (PHFs) in Alzheimer's disease (AD), can be characterised immunochemically by C-terminal truncation at Glu-391 recognised by monoclonal antibody (mAb) 423, and acid-reversible occlusion of a generic tau epitope in the tandem repeat region recognised by mAb 7.51. PHFs are also characterised by the presence of binding sites for a fluorescent dye (thiazin red) which can be used to differentiate between amorphous and fibrillar states of tau and beta-amyloid proteins in AD. We have used double labelling confocal microscopy to investigate that state of aggregation of the tau antigens associated with the core structure of the PHF at early stages of neurofibrillary pathology. We report that the early abnormal tau deposits in cells vulnerable to neurofibrillary degeneration are characterised by C-terminal truncation at Glu-391, acid-reversible occlusion of the mAb 7.51 epitope, and the absence of binding sites for thiazin red, consistent with the amorphous non-fibrillar structure demonstrated by immunoelectron microscopy. Transition to the fibrillar state in the PHF is associated with acid-reversible occlusion of both mAb 7.51 and 423 epitopes, and acquisition of binding sites for thiazin red. In neurites, the transition between the two states of aggregation shows distal to proximal polarity, with the fibrillar state found nearest the cell body. These findings demonstrate that the assembly of tau protein into the PHF occurs in at least two stages, an amorphous stage characterised by C-terminal truncation and occlusion of sites within the tandem repeat region, and a fibrillar stage characterised by acid-reversible occlusion of both epitopes via addition of intact tau molecules in the fuzzy coat of the PHF.
The phosphorylation state of tau changes during neurodevelopment and highly phosphorylated tau accumulates in the paired helical filaments found in Alzheimer's disease. In non-neuronal mammalian cells transiently expressed tau is predominantly not phosphorylated at sites known to be phosphorylated in paired helical filaments. However this pattern of phosphorylation is induced by both glycogen synthase kinase-3 alpha and -3 beta and here we show that this results in a change in the intracellular properties of tau. Within cells tau is bound to cytoskeletal structures and causes changes in cellular cytoarchitecture with the induction of thick and stable microtubule bundles. This morphology is lost when tau is co-expressed with glycogen synthase kinase-3 beta; microtubules become less stable and are not bound by tau. Independently of any direct or indirect effects on tau, glycogen synthase kinase-3 beta induces some but relatively slight changes in microtubule organization with the loss of a prominent centrosomal microtubular origin. The cytoskeleton is critical to cell function and within post-mitotic neurons has a highly specialized structure induced, in part, by the neuronal-specific microtubule-associated proteins such as tau. In vitro studies have suggested that the properties of tau are regulated by phosphorylation as highly phosphorylated tau does not promote tubulin polymer assembly. We have demonstrated, in intact cells, that tau highly phosphorylated in the presence of glycogen synthase kinase-3 beta loses the properties of microtubule binding and stabilization, suggesting that regulation of tau phosphorylation by this enzyme might be an important mechanism whereby cytoskeletal function is modulated during neurodevelopment and lost in neurodegeneration.
Huntington's disease (HD) is one of an increasing number of neurodegenerative disorders caused by a CAG/polyglutamine repeat expansion. Mice have been generated that are transgenic for the 5' end of the human HD gene carrying (CAG)115-(CAG)150 repeat expansions. In three lines, the transgene is ubiquitously expressed at both mRNA and protein level. Transgenic mice exhibit a progressive neurological phenotype that exhibits many of the features of HD, including choreiform-like movements, involuntary stereotypic movements, tremor, and epileptic seizures, as well as nonmovement disorder components. This transgenic model will greatly assist in an eventual understanding of the molecular pathology of HD and may open the way to the testing of intervention strategies.
Synelfin is a presynaptic protein of unknown function that is differentially regulated in the avian song control circuit during the critical period for song learning; in humans, it gives rise to an amyloidogenic peptide found in senile plaques of Alzheimer's disease. To gain insight into the potential involvement of synelfin in synapse development, we investigated its expression in neurons cultured from the embryonic rat hippocampus. These neurons express a variety of defined synaptic proteins, and form numerous synaptic connections after several days in culture. Synapsin I, a synaptic vesicle-associated protein, was detected within one day after the neurons were put in culture, but significant immunoreactivity for synelfin was not detected until approximately 5 days in vitro (DIV). By 3 DIV, synapsin-positive puncta (previously shown to correspond to presynaptic specializations) were detected surrounding the soma and proximal dendritic processes, whereas comparable aggregations of synelfin did not appear until several days later. By 14 DIV the punctate concentrations of synelfin and synapsin overlapped completely. Thus synelfin is expressed in these cultured neurons and eventually becomes localized to presynaptic terminals, but it is absent from these specializations when they first form. We conclude that presynaptic terminals can change in molecular composition, and that synelfin is associated with later stages in synaptic development or modulation.