肝性脑病患者脑脊液Tau 蛋白水平测定及其临床意义

目的:探讨肝性脑病患者脑脊液中总Tau蛋白(t-Tau)和磷酸化Tau蛋白(p-Tau)水平的变化及其与疾病严重程度的相关性。方法:采集26例肝性脑病患者及31例健康对照的脑脊液标本,采用酶联免疫吸附法(Elisa)检测脑脊液中的t-Tau和p-Tau水平,分析其与Child-Pugh评分和West Haven分级的相关性。结果:(1)肝性脑病患者脑脊液t-Tau和p-Tau水平显著高于健康对照组(P0.01)。(2)肝性脑病患者脑脊液t-Tau(γ=0.876,P0.01;γ=0.952,P0.01)、p-Tau(γ=0.808,P0.01;γ=0.808,P0.01)水平与Child-Pugh评分及West-Haven分级呈正相关。结论:由于脑脊液t-Tau和p-Tau水平为神经元损害的标志物,同时反应脑内应激状况,本研究证实肝性脑病患者脑内处于应激状态并有神经元损伤。     

The human MAPT locus generates circular RNAs

The microtubule-associated protein Tau, generated by the MAPT gene is involved in dozens of neurodegenerative conditions (“tauopathies”), including Alzheimer's disease (AD) and frontotemporal lobar degeneration/frontotemporal dementia (FTLD/FTD). The pre-mRNA of MAPT is well studied and its aberrant pre-mRNA splicing is associated with frontotemporal dementia. Using a PCR screen of RNA from human brain tissues, we found that the MAPT locus generates circular RNAs through a backsplicing mechanism from exon 12 to either exon 10 or 7. MAPT circular RNAs are localized in the cytosol and contain open reading frames encoding Tau protein fragments. The MAPT exon 10 is alternatively spliced and proteins involved in its regulation, such as CLK2, SRSF7/9G8, PP1 (protein phosphatase 1) and NIPP1 (nuclear inhibitor of PP1) reduce the abundance of the circular MAPT exon 12?→?10 backsplice RNA after being transfected into cultured HEK293 cells. In summary, we report the identification of new bona fide human brain RNAs produced from the MAPT locus. These may be a component of normal human brain Tau regulation and, since the circular RNAs could generate high molecular weight proteins with multiple microtubule binding sites, they could contribute to taupathies.     

Intracellular Clusterin Interacts with Brain Isoforms of the Bridging Integrator 1 and with the Microtubule-Associated Protein Tau in Alzheimer's Disease

Sporadic or late-onset Alzheimer''s disease (AD) is expected to affect 50% of individuals reaching 85 years of age. The most significant genetic risk factor for late-onset AD is the e4 allele of APOE gene encoding apolipoprotein E, a lipid carrier shown to modulate brain amyloid burden. Recent genome-wide association studies have uncovered additional single nucleotide polymorphisms (SNPs) linked to AD susceptibility, including those in the CLU and BIN1 genes encoding for clusterin (CLU) and the bridging integrator 1 (BIN1) proteins, respectively. Because CLU has been implicated in brain amyloid-β (Aβ) clearance in mouse models of amyloid deposition, we sought to investigate whether an AD-linked SNP in the CLU gene altered Aβ42 biomarker levels in the cerebrospinal fluid (CSF). Instead, we found that the CLU rs11136000 SNP modified CSF levels of the microtubule-associated protein Tau in AD patients. We also found that an intracellular form of CLU (iCLU) was upregulated in the brain of Tau overexpressing Tg4510 mice, but not in Tg2576 amyloid mouse model. By overexpressing iCLU and Tau in cell culture systems we discovered that iCLU was a Tau-interacting protein and that iCLU associated with brain-specific isoforms of BIN1, also recently identified as a Tau-binding protein. Through expression analysis of CLU and BIN1 variants, we found that CLU and BIN1 interacted via their coiled-coil motifs. In co-immunoprecipitation studies using human brain tissue, we showed that iCLU and the major BIN1 isoform expressed in neurons were associated with modified Tau species found in AD. Finally, we showed that expression of certain coding CLU variants linked to AD risk led to increased levels of iCLU. Together, our findings suggest that iCLU and BIN1 interaction might impact Tau function in neurons and uncover potential new mechanisms underlying the etiology of Tau pathology in AD.     

Propagation of Tau Misfolding from the Outside to the Inside of a Cell

Tauopathies are neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau in neurons and glia. Although Tau is normally considered an intracellular protein, Tau aggregates are observed in the extracellular space, and Tau peptide is readily detected in the cerebrospinal fluid of patients. Tau aggregation occurs in many diseases, including Alzheimer disease and frontotemporal dementia. Tau pathology begins in discrete, disease-specific regions but eventually involves much larger areas of the brain. It is unknown how this propagation of Tau misfolding occurs. We hypothesize that extracellular Tau aggregates can transmit a misfolded state from the outside to the inside of a cell, similar to prions. Here we show that extracellular Tau aggregates, but not monomer, are taken up by cultured cells. Internalized Tau aggregates displace tubulin, co-localize with dextran, a marker of fluid-phase endocytosis, and induce fibrillization of intracellular full-length Tau. These intracellular fibrils are competent to seed fibril formation of recombinant Tau monomer in vitro. Finally, we observed that newly aggregated intracellular Tau transfers between co-cultured cells. Our data indicate that Tau aggregates can propagate a fibrillar, misfolded state from the outside to the inside of a cell. This may have important implications for understanding how protein misfolding spreads through the brains of tauopathy patients, and it is potentially relevant to myriad neurodegenerative diseases associated with protein misfolding.Tau filament deposition in Alzheimer disease (AD),² frontotemporal dementia (FTD), and other tauopathies correlates closely with cognitive dysfunction and cell death (1). Mutations in the tau gene cause autosomal dominant tauopathy, implicating Tau as the proximal cause (2–4). Specific disease phenotypes are defined by the early sites of pathology. For example, AD is characterized by memory loss that derives from involvement of hippocampal neurons, whereas FTD is characterized by personality changes that result from frontal lobe involvement (5). Pathology ultimately spreads to involve much larger regions of brain. Studies on patients with AD show a progressive, stereotyped spread of Tau deposits from the transentorhinal cortex to the hippocampus, and eventually to most cortical areas (6–8). Others have correlated the distribution of neurofibrillary tangles of Tau in AD brains with trans-synaptic distance from the affected areas (9). A similar spread affecting different subsets of neurons has been observed in other sporadic tauopathies, such as progressive supranuclear palsy (10). It is unknown why Tau misfolding progresses through the brain, whether it is a sequence of cell autonomous processes or whether a toxic factor is involved. Loss of synaptic connections and cell death may expose healthy cells to toxic factors and decrease available neurotrophins (11, 12). Another possibility is that the Tau protein itself serves as the agent of trans-cellular propagation. For example, it has been shown that extracellular Tau is toxic to cultured neuronal cells (13, 14). This is consistent with the observation that immunotherapy against Tau reduces pathology in a mouse model (15).Tau is well known as an intracellular protein that stabilizes microtubule filaments (16); however, it is readily detected in cerebrospinal fluid (17) and as extracellular aggregates, termed “ghost tangles,” in diseased brain. These are comprised predominantly of the microtubule-binding region (MTBR), the functional and pathogenic core of the Tau protein (18). We hypothesize that Tau aggregates present in the extracellular space enter naive cells and induce misfolding of intracellular Tau. We have tested this idea using cellular studies, biochemistry, and atomic force microscopy (AFM).     

Tau aggregates improve the Purinergic receptor P2Y12-associated podosome rearrangements in microglial cells

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is associated with protein misfolding, plaque accumulation, neuronal dysfunction, synaptic loss, and cognitive decline. The pathological cascade of AD includes the intracellular Tau hyperphosphorylation and its subsequent aggregation, extracellular Amyloid-β plaque formation and microglia-mediated neuroinflammation. The extracellular release of aggregated Tau is sensed by surveilling microglia through the involvement of various cell surface receptors. Among all, purinergic P2Y12R signaling is involved in microglial chemotaxis towards the damaged neurons. Microglial migration is highly linked with membrane-associated actin remodeling leading to the phagocytosis of extracellular Tau species. Here, we studied the formation of various actin structures such as podosome, lamellipodia and filopodia, in response to extracellular Tau monomers and aggregates. Microglial podosomes are colocalized with actin nucleator protein WASP, Arp2 and TKS5 adaptor protein during Tau-mediated migration. Moreover, the P2Y12 receptors were associated with F-actin-rich podosome structures, which signify the potential of Tau aggregates in microglial chemotaxis through the involvement of actin remodeling.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

Bin1 antibody lowers the expression of phosphorylated Tau in Alzheimer's disease

Alzheimer's disease (AD) is an irreversible, progressive brain disorder responsible for memory loss leading to the inability to carry out the simplest tasks. AD is one of the leading causes of death in the United States. As yet there are no effective medications to treat this debilitating disease. In recent years, a human gene called bridging integrator 1 (BIN1) has emerged as one of the most important genes in affecting the incidence of sporadic AD. Bin1 can directly bind to Tau and mediates late onset AD risk by modulating Tau pathology. Recently our group found Bin1 antibody could exert drug-like properties in an animal model of ulcerative colitis. We hypothesized that the Bin1 monoclonal antibody (mAb) could be used in the treatment of AD by lowering the levels of Tau in cell culture and animal models. Cell culture studies confirmed that the Bin1 mAb (99D) could lower the levels of phosphorylated Tau (pTau). Multiple mechanisms aided by endosomal proteins and Fc gamma receptors are involved in the uptake of Bin1 mAb into cells. In Tau expressing cell culture, the Bin1 mAb induces the proteasome machinery leading to ubiquitination of molecules thereby preventing cell stress. In vivo studies demonstrated that treatment of P301S mice expressing Tau with the Bin1 mAb survived longer than the untreated mice. Our data confirm that Bin1 mAb lowers the levels of pTau and could be a drug candidate in the treatment of AD.     

Tau missorting and spastin-induced microtubule disruption in neurodegeneration: Alzheimer Disease and Hereditary Spastic Paraplegia

In Alzheimer Disease (AD), the mechanistic connection of the two major pathological hallmarks, namely deposition of Amyloid-beta (Aβ) in the form of extracellular plaques, and the pathological changes of the intracellular protein Tau (such as phosphorylation, missorting, aggregation), is not well understood. Genetic evidence from AD and Down Syndrome (Trisomy 21), and animal models thereof, suggests that aberrant production of Aβ is upstream of Tau aggregation, but also points to Tau as a critical effector in the pathological process. Yet, the cascade of events leading from increased levels of Aβ to Tau-dependent toxicity remains a matter of debate.Using primary neurons exposed to oligomeric forms of Aβ, we have found that Tau becomes mislocalized (missorted) into the somatodendritic compartment. Missorting of Tau correlates with loss of microtubules and downstream consequences such as loss of mature spines, loss of synaptic activity, and mislocalization of mitochondria.In this cascade, missorting of Tau induces mislocalization of TTLL6 (Tubulin-Tyrosine-Ligase-Like 6) into the dendrites. TTLL6 induces polyglutamylation of microtubules, which acts as a trigger for spastin mediated severing of dendritic microtubules. Loss of microtubules makes cells unable to maintain transport of mitochondria, which in turn results in synaptic dysfunction and loss of mature spines. These pathological changes are absent in TauKO derived primary neurons. Thus, Tau mediated mislocalization of TTLL6 and spastin activation reveals a pathological gain of function for Tau and spastin in this cellular model system of AD.In contrast, in hereditary spastic paraplegia (HSP) caused by mutations of the gene encoding spastin (spg4 alias SPAST), spastin function in terms of microtubule severing is decreased at least for the gene product of the mutated allele, resulting in overstable microtubules in disease model systems. Whether total spastin severing activity or microtubule stability in human disease is also affected is not yet clear. No human disease has been associated so far with the long-chain polyglutamylation enzyme TTLL6, or the other TTLLs (1,5,11) possibly involved.Here we review the findings supporting a role for Tau, spastin and TTLL6 in AD and other tauopathies, HSP and neurodegeneration, and summarize possible therapeutic approaches for AD and HSP.     

Tau Trimers Are the Minimal Propagation Unit Spontaneously Internalized to Seed Intracellular Aggregation

Tau amyloid assemblies propagate aggregation from the outside to the inside of a cell, which may mediate progression of the tauopathies. The critical size of Tau assemblies, or “seeds,” responsible for this activity is currently unknown, but this could be important for the design of effective therapies. We studied recombinant Tau repeat domain (RD) and Tau assemblies purified from Alzheimer disease (AD) brain composed largely of full-length Tau. Large RD fibrils were first sonicated to create a range of assembly sizes. We confirmed our ability to resolve stable assemblies ranging from n = 1 to >100 units of Tau using size exclusion chromatography, fluorescence correlation spectroscopy, cross-linking followed by Western blot, and mass spectrometry. All recombinant Tau assemblies bound heparan sulfate proteoglycans on the cell surface, which are required for Tau uptake and seeding, because they were equivalently sensitive to inhibition by heparin and chlorate. However, cells only internalized RD assemblies of n ≥ 3 units. We next analyzed Tau assemblies from AD or control brains. AD brains contained aggregated species, whereas normal brains had predominantly monomer, and no evidence of large assemblies. HEK293 cells and primary neurons spontaneously internalized Tau of n ≥ 3 units from AD brain in a heparin- and chlorate-sensitive manner. Only n ≥ 3-unit assemblies from AD brain spontaneously seeded intracellular Tau aggregation in HEK293 cells. These results indicate that a clear minimum size (n = 3) of Tau seed exists for spontaneous propagation of Tau aggregation from the outside to the inside of a cell, whereas many larger sizes of soluble aggregates trigger uptake and seeding.     

Trans-cellular propagation of Tau aggregation by fibrillar species

Aggregation of the microtubule associated protein Tau is associated with several neurodegenerative disorders, including Alzheimer disease and frontotemporal dementia. In Alzheimer disease, Tau pathology spreads progressively throughout the brain, possibly along existing neural networks. However, it is still unclear how the propagation of Tau misfolding occurs. Intriguingly, in animal models, vaccine-based therapies have reduced Tau and synuclein pathology by uncertain mechanisms, given that these proteins are intracellular. We have previously speculated that trans-cellular propagation of misfolding could be mediated by a process similar to prion pathogenesis, in which fibrillar Tau aggregates spread pathology from cell to cell. However, there has been little evidence to demonstrate true trans-cellular propagation of Tau misfolding, in which Tau aggregates from one cell directly contact Tau protein in the recipient cell to trigger further aggregation. Here we have observed that intracellular Tau fibrils are directly released into the medium and then taken up by co-cultured cells. Internalized Tau aggregates induce fibrillization of intracellular Tau in these naive recipient cells via direct protein-protein contact that we demonstrate using FRET. Tau aggregation can be amplified across several generations of cells. An anti-Tau monoclonal antibody blocks Tau aggregate propagation by trapping fibrils in the extracellular space and preventing their uptake. Thus, propagation of Tau protein misfolding among cells can be mediated by release and subsequent uptake of fibrils that directly contact native protein in recipient cells. These results support the model of aggregate propagation by templated conformational change and suggest a mechanism for vaccine-based therapies in neurodegenerative diseases.     

Seeded Aggregation and Toxicity of α-Synuclein and Tau: CELLULAR MODELS OF NEURODEGENERATIVE DISEASES*

The deposition of amyloid-like filaments in the brain is the central event in the pathogenesis of neurodegenerative diseases. Here we report cellular models of intracytoplasmic inclusions of α-synuclein, generated by introducing nucleation seeds into SH-SY5Y cells with a transfection reagent. Upon introduction of preformed seeds into cells overexpressing α-synuclein, abundant, highly filamentous α-synuclein-positive inclusions, which are extensively phosphorylated and ubiquitinated and partially thioflavin-positive, were formed within the cells. SH-SY5Y cells that formed such inclusions underwent cell death, which was blocked by small molecular compounds that inhibit β-sheet formation. Similar seed-dependent aggregation was observed in cells expressing four-repeat Tau by introducing four-repeat Tau fibrils but not three-repeat Tau fibrils or α-synuclein fibrils. No aggregate formation was observed in cells overexpressing three-repeat Tau upon treatment with four-repeat Tau fibrils. Our cellular models thus provide evidence of nucleation-dependent and protein-specific polymerization of intracellular amyloid-like proteins in cultured cells.     

A Novel Triple Repeat Mutant Tau Transgenic Model That Mimics Aspects of Pick’s Disease and Fronto-Temporal Tauopathies

Tauopathies are a group of disorders leading to cognitive and behavioral impairment in the aging population. While four-repeat (4R) Tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and Alzheimer’s disease, three-repeat (3R) Tau is the most abundant splice, in Pick''s disease. A number of transgenic models expressing wild-type and mutant forms of the 4R Tau have been developed. However, few models of three-repeat Tau are available. A transgenic mouse model expressing three-repeat Tau was developed bearing the mutations associated with familial forms of Pick''s disease (L266V and G272V mutations). Two lines expressing high (Line 13) and low (Line 2) levels of the three-repeat mutant Tau were analyzed. By Western blot, using antibodies specific to three-repeat Tau, Line 13 expressed 5-times more Tau than Line 2. The Tau expressed by these mice was most abundant in the frontal-temporal cortex and limbic system and was phosphorylated at residues detected by the PHF-1, AT8, CP9 and CP13 antibodies. The higher-expressing mice displayed hyperactivity, memory deficits in the water maze and alterations in the round beam. The behavioral deficits started at 6-8 months of age and were associated with a progressive increase in the accumulation of 3R Tau. By immunocytochemistry, mice from Line 13 displayed extensive accumulation of 3R Tau in neuronal cells bodies in the pyramidal neurons of the neocortex, CA1-3 regions, and dentate gyrus of the hippocampus. Aggregates in the granular cells had a globus appearance and mimic Pick’s-like inclusions. There were abundant dystrophic neurites, astrogliosis and synapto-dendritic damage in the neocortex and hippocampus of the higher expresser line. The hippocampal lesions were moderately argyrophilic and Thioflavin-S negative. By electron microscopy, discrete straight filament aggregates were detected in some neurons in the hippocampus. This model holds promise for better understanding the natural history and progression of 3R tauopathies and their relationship with mitochondrial alterations and might be suitable for therapeutical testing.     

BAG-1 associates with Hsc70.Tau complex and regulates the proteasomal degradation of Tau protein

Intraneuronal accumulation of phosphorylated Tau protein is a molecular pathology found in many forms of dementia, including Alzheimer disease. Research into possible mechanisms leading to the accumulation of modified Tau protein and the possibility of removing Tau protein from the system have revealed that the chaperone protein system can interact with Tau and mediate its degradation. Hsp70/Hsc70, a member of the chaperone protein family, interacts with Tau protein and mediates proper folding of Tau and can promote degradation of Tau protein under certain circumstances. However, because Hsp70/Hsc70 has many binding partners that can mediate its activity, there is still much to discover about how Hsp70 acts in vivo to regulate Tau protein. BAG-1, an Hsp70/Hsc70 binding partner, has been implicated as a mediator of neuronal function. In this work we show that BAG-1 associates with Tau protein in an Hsc70-dependent manner. Overexpression of BAG-1 induced an increase in Tau levels, which is shown to be due to an inhibition of protein degradation. We further show that BAG-1 can inhibit the degradation of Tau protein by the 20 S proteasome but does not affect the ubiquitination of Tau protein. RNA-mediated interference depletion of BAG-1 leads to a decrease in total Tau protein levels as well as promoting hyperphosphorylation of the remaining protein. Induction of Hsp70 by heat shock enhanced the increase of Tau levels in cells overexpressing BAG-1 but induced a decrease of Tau levels in cells that were depleted of BAG-1. Finally, BAG-1 is highly expressed in neurons bearing Tau tangles in a mouse model of Alzheimer disease. This data suggests a molecular mechanism through which Tau protein levels are regulated in the cell and possible consequences for the pathology and treatment of Alzheimer disease.     

Cerebrospinal Fluid PKR Level Predicts Cognitive Decline in Alzheimer’s Disease

The cerebrospinal fluid (CSF) levels of the proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR) are increased in Alzheimer’s disease (AD), but whether CSF PKR concentrations are associated with cognitive decline in AD patients remain unknown. In this study, 41 consecutive patients with AD and 11 patients with amnestic mild cognitive impairment (aMCI) from our Memory Clinic were included. A lumbar puncture was performed during the following month of the clinical diagnosis and Mini-Mental State Examination (MMSE) evaluations were repeated every 6 months during a mean follow-up of 2 years. In AD patients, linear mixed models adjusted for age and sex were used to assess the cross-sectional and longitudinal associations between MMSE scores and baseline CSF levels of Aβ peptide (Aβ 1-42), Tau, phosphorylated Tau (p-Tau 181), PKR and pPKR. The mean (SD) MMSE at baseline was 20.5 (6.1) and MMSE scores declined over the follow-up (-0.12 point/month, standard error [SE] = 0.03). A lower MMSE at baseline was associated with lower levels of CSF Aβ 1–42 and p-Tau 181/Tau ratio. pPKR level was associated with longitudinal MMSE changes over the follow-up, higher pPKR levels being related with an exacerbated cognitive deterioration. Other CSF biomarkers were not associated with MMSE changes over time. In aMCI patients, mean CSF biomarker levels were not different in patients who converted to AD from those who did not convert.These results suggest that at the time of AD diagnosis, a higher level of CSF pPKR can predict a faster rate of cognitive decline.     

Distinct Therapeutic Mechanisms of Tau Antibodies: PROMOTING MICROGLIAL CLEARANCE VERSUS BLOCKING NEURONAL UPTAKE*

Tauopathies are neurodegenerative diseases characterized by accumulation of Tau amyloids, and include Alzheimer disease and certain frontotemporal dementias. Trans-neuronal propagation of amyloid mediated by extracellular Tau may underlie disease progression. Consistent with this, active and passive vaccination studies in mouse models reduce pathology, although by unknown mechanisms. We previously reported that intracerebroventricular administration of three anti-Tau monoclonal antibodies (HJ8.5, HJ9.3, and HJ9.4) reduces pathology in a model overexpressing full-length mutant (P301S) human Tau. We now study effects of these three antibodies and a negative control antibody (HJ3.4) on Tau aggregate uptake into BV2 microglial-like cells and primary neurons. Antibody-independent Tau uptake into BV2 cells was blocked by heparin, consistent with a previously described role for heparan sulfate proteoglycans. Two therapeutic antibodies (HJ8.5 and HJ9.4) promoted uptake of full-length Tau fibrils into microglia via Fc receptors. Surprisingly, HJ9.3 promoted uptake of fibrils composed of the Tau repeat domain or Alzheimer disease-derived Tau aggregates, but failed to influence full-length recombinant Tau fibrils. Size fractionation of aggregates showed that antibodies preferentially promote uptake of larger oligomers (n ≥∼20-mer) versus smaller oligomers (n ∼10-mer) or monomer. No antibody inhibited uptake of full-length recombinant fibrils into primary neurons, but HJ9.3 blocked neuronal uptake of Tau repeat domain fibrils and Alzheimer disease-derived Tau. Antibodies thus have multiple potential mechanisms, including clearance via microglia and blockade of neuronal uptake. However these effects are epitope- and aggregate size-dependent. Establishing specific mechanisms of antibody activity in vitro may help in design and optimization of agents that are more effective in vivo.     

Neuropsychological and behavioural correlates of CSF biomarkers in dementia

To improve clinical, neuropsychological and behavioural characterisation of the cerebrospinal fluid (CSF) biomarkers beta-amyloid((1-42)) protein (Abeta42), protein tau (tau) and tau phosphorylated at threonine 181 (P-tau181) across diagnostic dementia categories, a prospective study was set up. Patients with probable Alzheimer's disease (AD) (n=201), AD with cerebrovascular disease (CVD) (AD+CVD) (n=33), frontotemporal dementia (FTD) (n=27), dementia with Lewy bodies (DLB) (n=22) and healthy controls (n=148) were included. All patients underwent neuropsychological examination and behavioural assessment by means of a battery of behavioural assessment scales. CSF was obtained by lumbar puncture and levels of Abeta42, tau and P-tau181 were determined with commercially available ELISA kits. Negative correlations between CSF Abeta42 levels and aggressiveness (Spearman: r=-0.223; p=0.002) and positive correlations with age at inclusion (r=0.195; p=0.006), age at onset (r=0.205; p=0.003) and MMSE scores (r=0.198; p=0.005) were found in AD. In AD+CVD, CSF Abeta42 levels were correlated with MMSE (r=0.482; p=0.006), Hierarchic Dementia Scale (r=0.503; p=0.017) and Boston Naming Test (r=0.516; p=0.012) scores. In controls, age was positively correlated with CSF tau (r=0.465; p<0.001) and P-tau181 levels (r=0.312; p<0.001). CSF tau and P-tau181 levels correlated significantly in all groups, whereas CSF Abeta42 correlated with tau and P-tau181 levels in healthy controls only. Negative correlations between CSF Abeta42 levels and aggressiveness were found in AD patients. CSF Abeta42 seems to be a stage marker for AD (+/-CVD) given the positive correlations with neuropsychological test results suggesting that CSF Abeta42 might be of help for monitoring disease progression. Different correlations between age and CSF biomarker levels were obtained in healthy controls compared to AD patients, indicating that AD-induced pathophysiological processes change age-dependent regulation of CSF biomarker levels.     

Hyperphosphorylation of Tau Induced by Naturally Secreted Amyloid-β at Nanomolar Concentrations Is Modulated by Insulin-dependent Akt-GSK3β Signaling Pathway

Alzheimer disease (AD) is neuropathologically characterized by the formation of senile plaques from amyloid-β (Aβ) and neurofibrillary tangles composed of phosphorylated Tau. Although there is growing evidence for the pathogenic role of soluble Aβ species in AD, the major question of how Aβ induces hyperphosphorylation of Tau remains unanswered. To address this question, we here developed a novel cell coculture system to assess the effect of extracellular Aβ at physiologically relevant levels naturally secreted from donor cells on the phosphorylation of Tau in recipient cells. Using this assay, we demonstrated that physiologically relevant levels of secreted Aβ are sufficient to cause hyperphosphorylation of Tau in recipient N2a cells expressing human Tau and in primary culture neurons. This hyperphosphorylation of Tau is inhibited by blocking Aβ production in donor cells. The expression of familial AD-linked PSEN1 mutants and APP ΔE693 mutant that induce the production of oligomeric Aβ in donor cells results in a similar hyperphosphorylation of Tau in recipient cells. The mechanism underlying the Aβ-induced Tau hyperphosphorylation is mediated by the impaired insulin signal transduction because we demonstrated that the phosphorylation of Akt and GSK3β upon insulin stimulation is less activated under this condition. Treating cells with the insulin-sensitizing drug rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, attenuates the Aβ-dependent hyperphosphorylation of Tau. These findings suggest that the disturbed insulin signaling cascade may be implicated in the pathways through which soluble Aβ induces Tau phosphorylation and further support the notion that correcting insulin signal dysregulation in AD may offer a potential therapeutic approach.     

Processing of neuropeptide Y, galanin, and somatostatin in the cerebrospinal fluid of patients with Alzheimer’s disease and frontotemporal dementia

Alzheimer's disease (AD) and frontotemporal dementia (FTD) are two prevalent neurodegenerative disorders for which the causes are unknown, except in rare familial cases. Several changes in neuropeptide levels as measured by radioimmunoassay (RIA) have been observed in these illnesses. Somatostatin (SOM) levels in cerebrospinal fluid (CSF) are consistently decreased in AD and FTD. Neuropeptide Y (NPY) levels are decreased in AD, but normal in FTD. Galanin (GAL) levels increase with the duration of illness in AD patients. The majority of studies of neuropeptides in CSF have not been verified by HPLC. The observed decrease in a neuropeptide level as measured by RIA may therefore reflect an altered synthesis or extracellular processing, resulting in neuropeptide fragments that may or may not be detected by RIA. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS) has been shown to be a powerful technique in the analysis of biological materials without any pre-treatment, by detecting peptides and proteins at a specific mass-to-charge (m/z) ratio. We studied the processing of the neuropeptides NPY, NPY, SOM and GAL in the cerebrospinal fluid of patients with AD (n = 3), FTD (n = 3) and controls (n = 2) using MALDI-MS. We found that considerable inter-individual variability exists in the rate of neuropeptide metabolism in CSF, as well as the number of peptide fragments formed. Certain patients showed differences in the processing of specific neuropeptides, relative to other patients and controls. This analysis of the metabolic processing of neuropeptides in CSF yielded a large amount of data for each individual studied. Further studies are required to determine the changes in neuropeptide processing that can be associated with AD and FTD. With further investigations using MALDI-MS analysis, it may be possible to identify a neuropeptide fragment or processing enzyme that can be correlated to these disease states.     

Somatodendritic accumulation of Tau in Alzheimer's disease is promoted by Fyn‐mediated local protein translation

The cause of protein accumulation in neurodegenerative disease is incompletely understood. In Alzheimer's disease (AD), the axonally enriched protein Tau forms hyperphosphorylated aggregates in the somatodendritic domain. Consequently, a process of subcellular relocalization driven by Tau phosphorylation and detachment from microtubules has been proposed. Here, we reveal an alternative mechanism of de novo protein synthesis of Tau and its hyperphosphorylation in the somatodendritic domain, induced by oligomeric amyloid‐β (Aβ) and mediated by the kinase Fyn that activates the ERK/S6 signaling pathway. Activation of this pathway is demonstrated in a range of cellular systems, and in vivo in brains from Aβ‐depositing, Aβ‐injected, and Fyn‐overexpressing mice with Tau accumulation. Both pharmacological inhibition and genetic deletion of Fyn abolish the Aβ‐induced Tau overexpression via ERK/S6 suppression. Together, these findings present a more cogent mechanism of Tau aggregation in disease. They identify a prominent role for neuronal Fyn in integrating signal transduction pathways that lead to the somatodendritic accumulation of Tau in AD.