Adeno-associated virus gene therapy with cholesterol 24-hydroxylase reduces the amyloid pathology before or after the onset of amyloid plaques in mouse models of Alzheimer's disease.

The development of Alzheimer's disease (AD) is closely connected with cholesterol metabolism. Cholesterol increases the production and deposition of amyloid-beta (Abeta) peptides that result in the formation of amyloid plaques, a hallmark of the pathology. In the brain, cholesterol is synthesized in situ but cannot be degraded nor cross the blood-brain barrier. The major exportable form of brain cholesterol is 24S-hydroxycholesterol, an oxysterol generated by the neuronal cholesterol 24-hydroxylase encoded by the CYP46A1 gene. We report that the injection of adeno-associated vector (AAV) encoding CYP46A1 in the cortex and hippocampus of APP23 mice before the onset of amyloid deposits markedly reduces Abeta peptides, amyloid deposits and trimeric oligomers at 12 months of age. The Morris water maze (MWM) procedure also demonstrated improvement of spatial memory at 6 months, before the onset of amyloid deposits. AAV5-wtCYP46A1 vector injection in the cortex and hippocampus of amyloid precursor protein/presenilin 1 (APP/PS) mice after the onset of amyloid deposits also reduced markedly the number of amyloid plaques in the hippocampus, and to a less extent in the cortex, 3 months after the injection. Our data demonstrate that neuronal overexpression of CYP46A1 before or after the onset of amyloid plaques significantly reduces Abeta pathology in mouse models of AD.

Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression.

Although the role of APP and PSEN genes in genetic Alzheimer's disease (AD) cases is well established, fairly little is known about the molecular mechanisms affecting Abeta generation in sporadic AD. Deficiency in Abeta clearance is certainly a possibility, but increased expression of proteins like APP or BACE1/beta-secretase may also be associated with the disease. We therefore investigated changes in microRNA (miRNA) expression profiles of sporadic AD patients and found that several miRNAs potentially involved in the regulation of APP and BACE1 expression appeared to be decreased in diseased brain. We show here that miR-29a, -29b-1, and -9 can regulate BACE1 expression in vitro. The miR-29a/b-1 cluster was significantly (and AD-dementia-specific) decreased in AD patients displaying abnormally high BACE1 protein. Similar correlations between expression of this cluster and BACE1 were found during brain development and in primary neuronal cultures. Finally, we provide evidence for a potential causal relationship between miR-29a/b-1 expression and Abeta generation in a cell culture model. We propose that loss of specific miRNAs can contribute to increased BACE1 and Abeta levels in sporadic AD.

MicroRNA regulation of Alzheimer's Amyloid precursor protein expression.

Gene dosage effects of Amyloid precursor protein (APP) can cause familial AD. Recent evidence suggest that microRNA (miRNA) pathways, implicated in gene transcriptional control, could be involved in the development of sporadic Alzheimer's disease (AD). We therefore investigated whether miRNAs could participate in the regulation of APP gene expression. We show that miRNAs belonging to the miR-20a family (that is, miR-20a, miR-17-5p and miR-106b) could regulate APP expression in vitro and at the endogenous level in neuronal cell lines. A tight correlation between these miRNAs and APP was found during brain development and in differentiating neurons. We thus identify miRNAs as novel endogenous regulators of APP expression, suggesting that variations in miRNA expression could contribute to changes in APP expression in the brain during development and disease. This possibility is further corroborated by the observation that a statistically significant decrease in miR-106b expression was found in sporadic AD patients.

Neuronal membrane cholesterol loss enhances amyloid peptide generation.

Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its beta-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

Biochemistry of Tau in Alzheimer's disease and related neurological disorders.

Microtubule-associated Tau proteins belong to a family of factors that polymerize tubulin dimers and stabilize microtubules. Tau is strongly expressed in neurons, localized in the axon and is essential for neuronal plasticity and network. From the very beginning of Tau discovery, proteomics methods have been essential to the knowledge of Tau biochemistry and biology. In this review, we have summarized the main contributions of several proteomic methods in the understanding of Tau, including expression, post-translational modifications and structure, in both physiological and pathophysiological aspects. Finally, recent advances in proteomics technology are essential to develop further therapeutic targets and early predictive and discriminative diagnostic assays for Alzheimer's disease and related disorders.

Tau pathology and neurodegeneration: an obvious but misunderstood link.

Most dementing disorders result from a degenerating process named tauopathy. Alzheimer disease is the most frequent one, but only one among the large spectrum of tau-related diseases. Cognitive impairment is related, first of all, to the neocortical location of this degenerating process. However, the nature and the mechanisms leading to tauopathy can be very different. This is demonstrated by familial mutations on the tau gene as well as by the different morphological and biochemical patterns of tau lesions. Therefore there is no doubt that tau is an etiological agent. But the persistent and unsolved question is the basic mechanism leading to neurodegeneration: is it due to the toxic effect of aggregated tau, or a loss of tau function, or both? Some answers may come from a more focused interest towards sporadic tauopathies. Most of them are characterized by a degenerating process starting in a specific and vulnerable brain area and consuming the connected neuronal network, like a chain reaction. In other words, sporadic tauopathies are mostly a destabilization of specific neuronal networks that should be modeled for an efficient therapeutic approach.

Research criteria for the diagnosis of Alzheimer's disease: revising the NINCDS-ADRDA criteria.

The NINCDS-ADRDA and the DSM-IV-TR criteria for Alzheimer's disease (AD) are the prevailing diagnostic standards in research; however, they have now fallen behind the unprecedented growth of scientific knowledge. Distinctive and reliable biomarkers of AD are now available through structural MRI, molecular neuroimaging with PET, and cerebrospinal fluid analyses. This progress provides the impetus for our proposal of revised diagnostic criteria for AD. Our framework was developed to capture both the earliest stages, before full-blown dementia, as well as the full spectrum of the illness. These new criteria are centred on a clinical core of early and significant episodic memory impairment. They stipulate that there must also be at least one or more abnormal biomarkers among structural neuroimaging with MRI, molecular neuroimaging with PET, and cerebrospinal fluid analysis of amyloid beta or tau proteins. The timeliness of these criteria is highlighted by the many drugs in development that are directed at changing pathogenesis, particularly at the production and clearance of amyloid beta as well as at the hyperphosphorylation state of tau. Validation studies in existing and prospective cohorts are needed to advance these criteria and optimise their sensitivity, specificity, and accuracy.

Absence of beta-amyloid deposits after immunization in Alzheimer disease with Lewy body dementia.

OBJECTIVE: To describe the neuropathological and biochemical findings of the brain examination of a patient enrolled in the AN-1792(QS-21) trial with an initial clinical diagnosis of Alzheimer disease (AD), in whom Lewy body variant was thereafter clinically diagnosed. DESIGN: A case report. SETTING: University memory clinic. Patient A 74-year-old woman with clinical features of probable AD. Intervention The patient received 2 injections of 225 mug of AN-1792 (beta-amyloid [Abeta]) plus 50 mug of the adjuvant QS-21 at an interval of 4 weeks. The patient was an antibody responder with an IgG anti-AN-1792 antibody titer exceeding 10 000 and an IgM titer exceeding 3500. Maximum serum anti-Abeta titers were reached in 4.7 months. During the 3 following years, while the Mini-Mental State Examination score remained globally stable despite several confusional episodes, she developed clinical features of dementia with Lewy bodies. The patient died 34 months postimmunization. An autopsy was performed. MAIN OUTCOME MEASURES: Neuropathological and biochemical examination of the brain using standardized evaluation for tau, beta-amyloid, and synuclein deposits. RESULTS: Neither neuropathological nor biochemical examinations showed amyloid deposit in the brain of this immunized patient. For tau deposition, Braak stage was IV/VI, and the Western blot analysis score was 9c/10. The neuropathological semiquantitative score for alpha-synuclein aggregation was 4. There was no inflammation. These results were compared with those of an age-matched patient with AD and a control devoid of any neurological disease. CONCLUSION: In this Lewy body variant case, with globally stable functional and cognitive features, Abeta immunization resulted in a significant clearance of amyloid deposits, with remaining tau and synuclein pathological features in the brain. Patients with a Lewy body variant of AD should not be excluded from enrollment in Abeta-immunization trials.

BRCA1 may modulate neuronal cell cycle re-entry in Alzheimer disease.

In Alzheimer disease, neuronal degeneration and the presence of neurofibrillary tangles correlate with the severity of cognitive decline. Neurofibrillary tangles contain the antigenic profile of many cell cycle markers, reflecting a re-entry into the cell cycle by affected neurons. However, while such a cell cycle re-entry phenotype is an early and consistent feature of Alzheimer disease, the mechanisms responsible for neuronal cell cycle are unclear. In this regard, given that a dysregulated cell cycle is a characteristic of cancer, we speculated that alterations in oncogenic proteins may play a role in neurodegeneration. To this end, in this study, we examined brain tissue from cases of Alzheimer disease for the presence of BRCA1, a known regulator of cell cycle, and found intense and specific localization of BRCA1 to neurofibrillary tangles, a hallmark lesion of the disease. Analysis of clinically normal aged brain tissue revealed systematically less BRCA1, and surprisingly in many cases with apparent phosphorylated tau-positive neurofibrillary tangles, BRCA1 was absent, yet BRCA1 was present in all cases of Alzheimer disease. These findings not only further define the cell cycle reentry phenotype in Alzheimer disease but also indicate that the neurofibrillary tangles which define Alzheimer disease may have a different genesis from the neurofibrillary tangles of normal aging.

Tau protein as a differential biomarker of tauopathies.

Microtubule-associated Tau proteins are the basic component of intraneuronal and glial inclusions observed in many neurological disorders, the so-called tauopathies. Many etiological factors, phosphorylation, splicing, and mutations, relate Tau proteins to neurodegeneration. Molecular analysis has revealed that hyperphosphorylation and abnormal phosphorylation might be one of the important events in the process leading to tau intracellular aggregation. Specific set of pathological tau proteins exhibiting a typical biochemical pattern, and a different regional and laminar distribution, could characterize five main classes of tauopathies. A direct correlation has been established between the regional brain distribution of tau pathology and clinical symptoms; for instance progressive involvement of neocortical areas is well correlated to the severity of dementia in Alzheimer's disease, overall suggesting that pathological tau proteins are reliable marker of the neurodegenerative process. Recent discovery of tau gene mutations in frontotemporal dementia with parkinsonism linked to chromosome 17 has reinforced the predominant role attributed to tau proteins in the pathogenesis of neurodegenerative disorders, and underlined the fact that distinct sets of tau isoforms expressed in different neuronal populations could lead to different pathologies. Overall, a better knowledge of the etiological factors responsible for the aggregation of tau proteins in brain diseases is essential for development of future differential diagnosis and therapeutic strategies. They would hopefully find their application against Alzheimer's disease but also in all neurological disorders for which a dysfunction of Tau biology has been identified.

The natural and molecular history of Alzheimer's disease.

Alzheimer's disease (AD) is a very frequent brain pathology of the elderly, with an etiology by far more complicated than thought in the nineties. In particular, the complexity comes from the coexistence of two degenerating processes, tau aggregation and Abeta deposition, that affect polymodal association brain areas, a feature never observed in non-human primates and difficult to model. Genetic studies have shown that AbetaPP plays a central role in familial and sporadic AD, but the role of tau has been for a long time understated. To apprehend this role, we have developed a spatio-temporal analysis of tauopathy in many brain areas of hundreds of non-demented and demented patients. This prospective and multidisciplinary study showed us that tauopathy always progresses in the brain along a very precise and invariable pathway, from the entorhinal then hippocampal formation to polymodal association areas to end in primary regions and in many subcortical areas. The cognitive impairment follows exactly the progression of the affected brain regions. In strict parallelism, neocortical Abeta deposits increase in quantity and heterogeneity, suggesting a direct link between both neurodegenerative processes. Altogether, our molecular study suggests that AD is a tauopathy fueled by AbetaPP dysfunction. Restoring AbetaPP loss of function seems to be the most efficient therapeutic approach.

[Tauopathy and Alzheimer disease: a full degenerating process]. / Tauopathie et maladie d'Alzheimer, un processus dégénératif à part entière.

Neurofibrillary degeneration is well correlated to the clinical signs of Alzheimer disease. However, the amyloid cascade is so well established in the scientific and medical community that the role of neurofibrillary degeneration in Alzheimer's disease etiopathogenesis is often underestimated. However, neuronal vulnerability is clearly a key factor for facilitating the amyloid pathology which allows the propagation of the degenerating process. In the present work, the role of tau pathology as both diagnostic marker and therapeutic target is highlighted in Alzheimer disease and related disorders.

Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins--a role in nuclear localization.

Both phosphorylation and O-GlcNAc glycosylation posttranslationally modify microtubule-associated Tau proteins. Whereas the hyperphosphorylation of these proteins that occurs in Alzheimer's disease is well characterized, little is known about the O-GlcNAc glycosylation. The present study demonstrates that a balance exists between phosphorylation and O-GlcNAc glycosylation of Tau proteins, and furthermore that a dysfunction of this balance correlates with reduced nuclear localization. The affinity of Tau proteins for WGA lectin, together with evidence from [3H]-galactose transfer and analysis of beta-eliminated products, demonstrated the presence of O-GlcNAc residues on both cytosolic and nuclear Tau proteins. In addition, our data indicated the existence of a balance between phosphorylation and O-GlcNAc glycosylation events. Indeed, as demonstrated by 2D-electrophoresis and Western blotting, O-GlcNAc residues were mainly located on the less phosphorylated Tau 441 variants, whereas the more phosphorylated forms were devoid of O-GlcNAc residues. Furthermore, the Tau protein hyperphosphorylation induced by cellular okadaic acid treatment was correlated with reduced incorporation of O-GlcNAc residues into Tau proteins and with diminished Tau transfer into the nucleus. Hence, this paper establishes a direct relationship between O-GlcNAc glycosylation, phosphorylation and cellular localization of Tau proteins.

A non-DM1, non-DM2 multisystem myotonic disorder with frontotemporal dementia: phenotype and suggestive mapping of the DM3 locus to chromosome 15q21-24.

The majority of proximal myotonic myopathy syndromes reported so far have been related to the myotonic dystrophy (DM) type 2 (DM2) mutation, an expanded (CCTG)n repeat in the ZNF9 gene. Here, we describe the phenotype and the histological features in muscle and brain of the first large pedigree with a non-myotonic dystrophy type 1 (DM1) non-DM2 multisystem myotonic disorder associated with severe frontotemporal dementia. Thirty individuals from three generations underwent detailed neurological, neuropsychological, electrophysiological, brain imaging and molecular analyses. Ten of them had proximal muscle weakness at onset, clinical/electrical myotonia and DM-type cataracts. The mean age at onset was 46.7 +/- 12.6 years (range: 32-69). Dementia was observed later in the course of the disease. On muscle biopsies, rare nuclear clumps, rimmed vacuoles and small angulated type 1 and type 2 fibres were seen early in the disease. They were replaced by fibrous adipose tissue at later stages. Immunohistochemical analysis of myosin heavy chain isoforms showed no selective fibre type atrophy-both type 1 and type 2 fibres being affected. Cortical atrophy without white matter lesions was seen on brain MRI. A brain single photon emission computed tomography (SPECT) study revealed marked frontotemporal hypoperfusion. Post-mortem examination of the brains of two patients showing prominent frontotemporal spongiosis, neuronal loss and rare neuronal and glial tau inclusions suggested frontotemporal dementia. Western blot analyses of the tau protein showed a triplet of isoforms (60, 64 and 69 kDa) in neocortical areas, and a doublet (64 and 69 kDa) in subcortical areas that distinguish our myotonic disorder from other's myotonic dystrophies. Molecular analyses failed to detect a repeat expansion in the DMPK and ZNF9 genes excluding both DM1 and DM2, whereas a genome-wide linkage analysis strongly suggested a linkage to chromosome 15q21-24. This previously unreported multisystem myotonic disorder including findings resembling DM1, DM2 and frontotemporal dementia provides further evidence of the clinical and genetic heterogeneity of the myotonic dystrophies. We propose to designate this disease myotonic dystrophy type 3, DM3.

Tauopathies: recent insights into old diseases.

Neurofibrillary tangles are brain lesions that have been discovered at the beginning of the 20th century, using histological silver staining. Tangles are intra-neuronal hallmarks of a degenerating process: neurofibrillary degeneration (NFD). The basic component involved in tangle formation is tau protein. Tangles are found in more than 20 different neurodegenerative disorders, suggesting that NFD is a unique consequence to different types of etiological factors. However, tangles have a morphological and biochemical signature which is disease-specific. They are made up of different types of filaments such as paired helical filaments (PHFs) in Alzheimer's disease or straight filaments in progressive supranuclear palsy. Tau aggregates have a disease-specific biochemical bar-code due to the aggregation of specific sets of tau isoforms. Tau lesions have also a disease-specific pattern of spatio-temporal progression in the human brain which is well correlated to cognitive impairment. At last, pathological tau mutations are at the origin of familial fronto-temporal diseases with parkinsonism (FTDP-17). Together, these observations have generated the concept of tauopathies. Indeed, each tauopathy is defined by a combination of clinical, neuropathological, biochemical and genetic features. Most of them have a specific defect on tau (mutation, aberrant splicing, abnormal phosphorylation, abnormal processing, neuronal or genotypic vulnerability), suggesting that, in fact, the etiology of most tauopathies is directly linked to tau dysfunction. In conclusion, we observe that most dementing disorders are tauopathies and that most demented patients have a tauopathy.

[Animal models of Alzheimer's disease: a road full of pitfalls]. / Modélisation de la maladie d'Alzheimer: un parcours semé d'embûches.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects people slowly, insidiously, progressively but irreversibly. This disease will destroy, little by little, the neurons of the hippocampal formation that sustain episodic memory, and the neurons of the polymodal association areas involved in all other cognitive functions. AD is characterized by two types of brain lesions: amyloid plaques and neurofibrillary degeneration. Three major molecular actors are involved in the dynamic of neurodegeneration, but the precise role of each is still a matter of debate: the first one is APP (amyloid protein precursor) that is cleaved to release Abeta peptide that will aggregate into plaques. The last one is the microtubule-associated protein tau that assembles into paired helical filaments in neurons to constitute neurofibrillary degeneration. The main difficulty to study AD results from the fact that this disease is specific to humans and, therefore, that there is no relevant animal model at our disposal. Transgenic mice merely reflect partial aspects of the physiopathological process, impeding therapeutic approaches such as relevant drug tests on animals. But research is in progress...

Chloroquine and chloroquinoline derivatives as models for the design of modulators of amyloid Peptide precursor metabolism.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). Preventing deregulated APP processing by inhibiting amyloidogenic processing of carboxy-terminal fragments (APP-CTFs), and reducing the toxic effect of amyloid beta (Aß) peptides remain an effective therapeutic strategy. We report the design of piperazine-containing compounds derived from chloroquine structure and evaluation of their effects on APP metabolism and ability to modulate the processing of APP-CTF and the production of Aß peptide. Compounds which retained alkaline properties and high affinity for acidic cell compartments were the most effective. The present study demonstrates that (1) the amino side chain of chloroquine can be efficiently substituted by a bis(alkylamino)piperazine chain, (2) the quinoline nucleus can be replaced by a benzyl or a benzimidazole moiety, and (3) pharmacomodulation of the chemical structure allows the redirection of APP metabolism toward a decrease of Aß peptide release, and increased stability of APP-CTFs and amyloid intracellular fragment. Moreover, the benzimidazole compound 29 increases APP-CTFs in vivo and shows promising activity by the oral route. Together, this family of compounds retains a lysosomotropic activity which inhibits lysosome-related Aß production, and is likely to be beneficial for therapeutic applications in AD.

Neurosteroid quantification in human brain regions: comparison between Alzheimer's and nondemented patients.

Some neurosteroids have been shown to display beneficial effects on neuroprotection in rodents. To investigate the physiopathological significance of neurosteroids in Alzheimer's disease (AD), we compared the concentrations of pregnenolone, pregnenolone sulfate (PREGS), dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEAS), progesterone, and allopregnanolone, measured by gas chromatography-mass spectrometry, in individual brain regions of AD patients and aged nondemented controls, including hippocampus, amygdala, frontal cortex, striatum, hypothalamus, and cerebellum. A general trend toward decreased levels of all steroids was observed in all AD patients' brain regions compared with controls: PREGS and DHEAS were significantly lower in the striatum and cerebellum, and DHEAS was also significantly reduced in the hypothalamus. A significant negative correlation was found between the levels of cortical beta-amyloid peptides and those of PREGS in the striatum and cerebellum and between the levels of phosphorylated tau proteins and DHEAS in the hypothalamus. This study provides reference values for steroid concentrations determined by gas chromatography-mass spectrometry in various regions of the aged human brain. High levels of key proteins implicated in the formation of plaques and neurofibrillary tangles were correlated with decreased brain levels of PREGS and DHEAS, suggesting a possible neuroprotective role of these neurosteroids in AD.

In vitro models of age-related neurodegenerative disorders.

Aging is the major risk factor for numerous brain diseases. This is especially true for Alzheimer's disease (AD), a peculiar neurodegenerative disorder in that it results from the synergy of two simultaneous but distinct degenerating processes: A beta and tau pathologies. For AD, and for most neurodegenerative disorders, aggregation of full length or truncated proteins, in neurons or glial cells, or in the parenchyma, is central, but still a mystery. In addition, the late onset of these pathologies links them to ageing processes. Cause or consequence? Experimental models, that allow us to dissect these pathophysiological defects, are presented.

Tau aggregation in the hippocampal formation: an ageing or a pathological process?

Tauopathy is a concept to describe different genetic or metabolic dysfunctions of tau proteins that generate most of the known dementing disorders. Tauopathy is a degenerating process that also affects the entorhinal formation, and then the hippocampal formation in ageing. In Alzheimer's disease (AD), a disease due to APP dysfunction, a similar tauopathy process in observed in neocortical areas, well correlated to cognitive impairment. One important gap of knowledge is the relationship between tauopathy in the hippocampal formation, ageing, AD, and cognitive impairment. Here we show that the multidisciplinary analysis of numerous brains from non-demented and demented patients suggests the following observations: tauopathy of the hippocampal formation in humans is age-related but not an age-dependent process, also independent of AD, but amplified by APP dysfunctions. Tauopathy in the entorhinal and hippocampal formation could be another type of pathological dysfunction of tau proteins, and a therapeutic target to delay AD. Relevant animal models are desperately needed to address this issue.