An in situ and in vitro investigation of cytoplasmic TDP-43 inclusions reveals the absence of a clear amyloid signature.

Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-ß-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology.Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ. We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions.Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-ß structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature.Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.KEY MESSAGESCytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-ß structure, and do not show a fibrillar morphology.TDP-43 assemblies formed in vitro from pure TDP-43 do not have any hallmarks of amyloid.

Stimulation of synaptic activity promotes TFEB-mediated clearance of pathological MAPT/Tau in cellular and mouse models of tauopathies.

Synapses represent an important target of Alzheimer disease (AD), and alterations of their excitability are among the earliest changes associated with AD development. Synaptic activation has been shown to be protective in models of AD, and deep brain stimulation (DBS), a surgical strategy that modulates neuronal activity to treat neurological and psychiatric disorders, produced positive effects in AD patients. However, the molecular mechanisms underlying the protective role(s) of brain stimulation are still elusive. We have previously demonstrated that induction of synaptic activity exerts protection in mouse models of AD and frontotemporal dementia (FTD) by enhancing the macroautophagy/autophagy flux and lysosomal degradation of pathological MAPT/Tau. We now provide evidence that TFEB (transcription factor EB), a master regulator of lysosomal biogenesis and autophagy, is a key mediator of this cellular response. In cultured primary neurons from FTD-transgenic mice, synaptic stimulation inhibits MTORC1 signaling, thus promoting nuclear translocation of TFEB, which, in turn, induces clearance of MAPT/Tau oligomers. Conversely, synaptic activation fails to promote clearance of toxic MAPT/Tau in neurons expressing constitutively active RRAG GTPases, which sequester TFEB in the cytosol, or upon TFEB depletion. Activation of TFEB is also confirmed in vivo in DBS-stimulated AD mice. We also demonstrate that DBS reduces pathological MAPT/Tau and promotes neuroprotection in Parkinson disease patients with tauopathy. Altogether our findings indicate that stimulation of synaptic activity promotes TFEB-mediated clearance of pathological MAPT/Tau. This mechanism, underlying the protective effect of DBS, provides encouraging support for the use of synaptic stimulation as a therapeutic treatment against tauopathies.Abbreviations: 3xTg-AD: triple transgenic AD mice; AD: Alzheimer disease; CSA: cyclosporine A; DBS: deep brain stimulation; DIV: days in vitro; EC: entorhinal cortex; FTD: frontotemporal dementia; gLTP: glycine-induced long-term potentiation; GPi: internal segment of the globus pallidus; PD: Parkinson disease; STN: subthalamic nucleus; TFEB: transcription factor EB.

Chronic deep brain stimulation in an Alzheimer's disease mouse model enhances memory and reduces pathological hallmarks.

BACKGROUND: Alzheimer's disease (AD) is a progressive degenerative disorder that currently remains extremely disabling. Recent work has shown that deep brain stimulation (DBS) has promising effects in AD patients. In parallel to the clinical trials, we investigated the impact of chronic DBS in 3xTg mice, a well-established animal model of AD. METHODS: AD mice were assigned to control (Cont), non-stimulation (NS) and stimulation (DBS) groups, along with age matched wild type controls (WT-Cont). Bilateral electrodes were implanted in the entorhinal cortex to deliver chronic high frequency stimulation for 25 days. Animals were tested in memory behavioral tasks, with post-mortem measurements of pathological markers. RESULTS: We found that chronic DBS in AD mice normalized their impaired performance in the Morris water maze task to that of the WT group in the probe test. In the novel object and novel place preference tasks, AD-DBS mice spent more time at the novel object and novice location compared to AD-NS mice. These cognitive improvements in AD-DBS mice were associated with DBS induced increased neurogenesis in the dentate gyrus, a significant reduction in ß-amyloid plaques, a reduction in CA-1 cellular ß-amyloid-42 levels, decreased cortical total-tau and phosphorylated-tau, along with decreased hippocampal total-tau. CONCLUSION: Overall, we show that chronic DBS of the entorhinal cortex in AD mice improves both memory and AD specific pathological markers. These results support further testing of DBS as a potential treatment in AD patients.

Synaptic activity and Alzheimer's disease: a critical update.

Synapses have been known for many years to be the crucial target of pathology in different forms of dementia, in particular Alzheimer's disease (AD). Synapses and their appropriate activation or inhibition are fundamental for the proper brain function. Alterations in synaptic/neuronal activity and brain metabolism are considered among the earliest symptoms linked to the progression of AD, and lead to a central question in AD research: what is the role played by synaptic activity in AD pathogenesis? Intriguingly, in the last decade, important studies demonstrated that the state of activation of synapses affects the homeostasis of beta-amyloid (Aß) and tau, both of which aggregate and accumulate during AD, and are involved in neuronal dysfunction. In this review we aim to summarize the up-to-date data linking synaptic/neuronal activity with Aß and tau; moreover, we also intend to provide a critical overview on brain activity alterations in AD, and their role in the disease's pathophysiology.

Methylene blue upregulates Nrf2/ARE genes and prevents tau-related neurotoxicity.

Methylene blue (MB, methylthioninium chloride) is a phenothiazine that crosses the blood brain barrier and acts as a redox cycler. Among its beneficial properties are its abilities to act as an antioxidant, to reduce tau protein aggregation and to improve energy metabolism. These actions are of particular interest for the treatment of neurodegenerative diseases with tau protein aggregates known as tauopathies. The present study examined the effects of MB in the P301S mouse model of tauopathy. Both 4 mg/kg MB (low dose) and 40 mg/kg MB (high dose) were administered in the diet ad libitum from 1 to 10 months of age. We assessed behavior, tau pathology, oxidative damage, inflammation and numbers of mitochondria. MB improved the behavioral abnormalities and reduced tau pathology, inflammation and oxidative damage in the P301S mice. These beneficial effects were associated with increased expression of genes regulated by NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE), which play an important role in antioxidant defenses, preventing protein aggregation, and reducing inflammation. The activation of Nrf2/ARE genes is neuroprotective in other transgenic mouse models of neurodegenerative diseases and it appears to be an important mediator of the neuroprotective effects of MB in P301S mice. Moreover, we used Nrf2 knock out fibroblasts to show that the upregulation of Nrf2/ARE genes by MB is Nrf2 dependent and not due to secondary effects of the compound. These findings provide further evidence that MB has important neuroprotective effects that may be beneficial in the treatment of human neurodegenerative diseases with tau pathology.

PGC-1α overexpression exacerbates ß-amyloid and tau deposition in a transgenic mouse model of Alzheimer's disease.

The peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) interacts with various transcription factors involved in energy metabolism and in the regulation of mitochondrial biogenesis. PGC-1α mRNA levels are reduced in a number of neurodegenerative diseases and contribute to disease pathogenesis, since increased levels ameliorate behavioral defects and neuropathology of Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. PGC-1α and its downstream targets are reduced both in postmortem brain tissue of patients with Alzheimer's disease (AD) and in transgenic mouse models of AD. Therefore, we investigated whether increased expression of PGC-1α would exert beneficial effects in the Tg19959 transgenic mouse model of AD; Tg19959 mice express the human amyloid precursor gene (APP) with 2 familial AD mutations and develop increased ß-amyloid levels, plaque deposition, and memory deficits by 2-3 mo of age. Rather than an improvement, the cross of the Tg19959 mice with mice overexpressing human PGC-1α exacerbated amyloid and tau accumulation. This was accompanied by an impairment of proteasome activity. PGC-1α overexpression induced mitochondrial abnormalities, neuronal cell death, and an exacerbation of behavioral hyperactivity in the Tg19959 mice. These findings show that PGC-1α overexpression exacerbates the neuropathological and behavioral deficits that occur in transgenic mice with mutations in APP that are associated with human AD.

Bezafibrate administration improves behavioral deficits and tau pathology in P301S mice.

Peroxisome proliferator-activated receptors (PPARs) are ligand-mediated transcription factors, which control both lipid and energy metabolism and inflammation pathways. PPARγ agonists are effective in the treatment of metabolic diseases and, more recently, neurodegenerative diseases, in which they show promising neuroprotective effects. We studied the effects of the pan-PPAR agonist bezafibrate on tau pathology, inflammation, lipid metabolism and behavior in transgenic mice with the P301S human tau mutation, which causes familial frontotemporal lobar degeneration. Bezafibrate treatment significantly decreased tau hyperphosphorylation using AT8 staining and the number of MC1-positive neurons. Bezafibrate treatment also diminished microglial activation and expression of both inducible nitric oxide synthase and cyclooxygenase 2. Additionally, the drug differentially affected the brain and brown fat lipidome of control and P301S mice, preventing lipid vacuoles in brown fat. These effects were associated with behavioral improvement, as evidenced by reduced hyperactivity and disinhibition in the P301S mice. Bezafibrate therefore exerts neuroprotective effects in a mouse model of tauopathy, as shown by decreased tau pathology and behavioral improvement. Since bezafibrate was given to the mice before tau pathology had developed, our data suggest that bezafibrate exerts a preventive effect on both tau pathology and its behavioral consequences. Bezafibrate is therefore a promising agent for the treatment of neurodegenerative diseases associated with tau pathology.

Critical role of intraneuronal Aß in Alzheimer's disease: technical challenges in studying intracellular Aß.

AIMS: Multiple lines of evidence have implicated ß-amyloid (Aß) in the pathogenesis of Alzheimer's disease (AD). However, the mechanism(s) whereby Aß is involved in the disease process remains unclear. The dominant hypothesis in AD has been that Aß initiates the disease via toxicity from secreted, extracellular Aß aggregates. More recently, an alternative hypothesis has emerged focusing on a pool of Aß that accumulates early on within AD vulnerable neurons of the brain. Although the topic of intraneuronal Aß has been of major interest in the field, technical difficulties in detecting intraneuronal Aß have also made this topic remarkably controversial. Here we review evidence pointing to the critical role of intraneuronal Aß in AD and provide insights both into challenges faced in detecting intracellular Aß and the prion-like properties of Aß. MAIN METHODS: Immunoprecipitation and Western blot are used for Aß detection. KEY FINDINGS: We highlight that a standard biochemical method can underestimate intraneuronal Aß and that extracellular Aß can up-regulate intracellular Aß. We also show that detergent can remove intraneuronal Aß. SIGNIFICANCE: There is a growing awareness that intraneuronal Aß is a key pathogenic pool of Aß involved in causing synapse dysfunction. Difficulties in detecting intraneuronal Aß are an insufficient reason for ignoring this critical pool of Aß.

Intraneuronal Aß accumulation, amyloid plaques, and synapse pathology in Alzheimer's disease.

BACKGROUND: ß-Amyloid (Aß) plaques are a pathological hallmark of Alzheimer's disease (AD) and multiple lines of evidence have linked Aß with AD. However, synapse loss is known as the best pathological correlate of cognitive impairment in AD, and intraneuronal Aß accumulation has been shown to precede plaque pathology. The progression of Aß accumulation to synapse loss and plaque formation remains incomplete. The objective is to investigate the progression of intraneuronal Aß accumulation in the brain. METHODS: To visualize and analyze the development of Aß pathology we perform immunohistochemistry and immunofluorescence microscopy using antibodies against different Aß conformations, synaptic proteins and structural neuronal proteins in brain tissue of AD transgenic mouse models. RESULTS: Our results show the intraneuronal onset of Aß42 accumulation in AD mouse brains with aging. We observe an inverse correlation of Aß and amyloid fibrils with structural proteins within neurites. Images reveal aggregated amyloid within selective pyramidal neurons, neurites and synapses in AD transgenic mice as plaques arise. CONCLUSION: The data support that Aß42 accumulation and aggregation begin within AD-vulnerable neurons in the brain. Progressive intraneuronal Aß42 aggregation disrupts the normal cytoarchitecture of neurites.

Impaired ß-amyloid secretion in Alzheimer's disease pathogenesis.

A central question in Alzheimer's disease (AD) research is what role ß-amyloid peptide (Aß) plays in synaptic dysfunction. Synaptic activity increases Aß secretion, potentially inhibiting synapses, but also decreases intraneuronal Aß, protecting synapses. We now show that levels of secreted Aß fall with time in culture in neurons of AD-transgenic mice, but not wild-type mice. Moreover, the ability of synaptic activity to elevate secreted Aß and reduce intraneuronal Aß becomes impaired in AD-transgenic but not wild-type neurons with time in culture. We demonstrate that synaptic activity promotes an increase in the Aß-degrading protease neprilysin at the cell surface and a concomitant increase in colocalization with Aß42. Remarkably, AD-transgenic but not wild-type neurons show reduced levels of neprilysin with time in culture. This impaired ability to secrete Aß and reduce intraneuronal Aß has important implications for the pathogenesis and treatment of AD.

High-resolution 3D reconstruction reveals intra-synaptic amyloid fibrils.

ß-Amyloid (Aß) accumulation and aggregation are hallmarks of Alzheimer's disease (AD). High-resolution three-dimensional (HR-3D) volumetric imaging allows for better analysis of fluorescence confocal microscopy and 3D visualization of Aß pathology in brain. Early intraneuronal Aß pathology was studied in AD transgenic mouse brains by HR-3D volumetric imaging. To better visualize and analyze the development of Aß pathology, thioflavin S staining and immunofluorescence using antibodies against Aß, fibrillar Aß, and structural and synaptic neuronal proteins were performed in the brain tissue of Tg19959, wild-type, and Tg19959-YFP mice at different ages. Images obtained by confocal microscopy were reconstructed into three-dimensional volumetric datasets. Such volumetric imaging of CA1 hippocampus of AD transgenic mice showed intraneuronal onset of Aß42 accumulation and fibrillization within cell bodies, neurites, and synapses before plaque formation. Notably, early fibrillar Aß was evident within individual synaptic compartments, where it was associated with abnormal morphology. In dendrites, increasing intraneuronal thioflavin S correlated with decreases in neurofilament marker SMI32. Fibrillar Aß aggregates could be seen piercing the cell membrane. These data support that Aß fibrillization begins within AD vulnerable neurons, leading to disruption of cytoarchitecture and degeneration of spines and neurites. Thus, HR-3D volumetric image analysis allows for better visualization of intraneuronal Aß pathology and provides new insights into plaque formation in AD.

Analysis of vesicular trafficking in primary neurons by live imaging.

Alzheimer's disease, the most common neurodegenerative disease, is characterized by a progressive loss of synapses and accumulation of amyloid-beta (Aß) peptides in the brain. Previous studies demonstrated that acute increase in synaptic activity in cultured hippocampal slices and mouse brains (Cirrito et al. Neuron 48: 913-922, 2005; Kamenetz et al. Neuron 37: 925-937, 2003) enhanced secretion of Aß. Since synaptic activity promotes Aß secretion, it could also affect the trafficking and processing of its precursor, the amyloid precursor protein (APP). Here, we describe a method to investigate the effect of acute synaptic activation on APP trafficking within dendrites.

Effects of synaptic modulation on beta-amyloid, synaptophysin, and memory performance in Alzheimer's disease transgenic mice.

Accumulation of ß-amyloid (Aß) and loss of synapses are hallmarks of Alzheimer's disease (AD). How synaptic activity relates to Aß accumulation and loss of synapses is a current topic of major interest. Synaptic activation promotes Aß secretion, and chronic reduction of synaptic activity reduced Aß plaques in an AD transgenic mouse model. This suggested beneficial effects of reducing synaptic activity in AD. We now show that reduced synaptic activity causes detrimental effects on synapses and memory despite reducing plaques using two different models of chronic synaptic inhibition: deafferentation of the barrel cortex and administration of benzodiazepine. An interval of prolonged synaptic inhibition exacerbated loss of synaptophysin compared with synaptically more active brain in AD transgenic but not wild-type mice. Furthermore, an interval of benzodiazepine treatment, followed by a washout period, exacerbated memory impairment in AD transgenic mice. Exacerbation of synaptic and behavioral abnormalities occurred in the setting of reduced Aß plaques but elevated intraneuronal Aß immunoreactivity. These data support beneficial effects of synaptic activation on Aß-related synaptic and behavioral impairment in AD.

Dysregulation of the mTOR pathway mediates impairment of synaptic plasticity in a mouse model of Alzheimer's disease.

BACKGROUND: The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr protein kinase that plays a pivotal role in multiple fundamental biological processes, including synaptic plasticity. We explored the relationship between the mTOR pathway and ß-amyloid (Aß)-induced synaptic dysfunction, which is considered to be critical in the pathogenesis of Alzheimer's disease (AD). METHODOLOGY/PRINCIPAL FINDINGS: We provide evidence that inhibition of mTOR signaling correlates with impairment in synaptic plasticity in hippocampal slices from an AD mouse model and in wild-type slices exposed to exogenous Aß1-42. Importantly, by up-regulating mTOR signaling, glycogen synthase kinase 3 (GSK3) inhibitors rescued LTP in the AD mouse model, and genetic deletion of FK506-binding protein 12 (FKBP12) prevented Aß-induced impairment in long-term potentiation (LTP). In addition, confocal microscopy demonstrated co-localization of intraneuronal Aß42 with mTOR. CONCLUSIONS/SIGNIFICANCE: These data support the notion that the mTOR pathway modulates Aß-related synaptic dysfunction in AD.

Synapses, synaptic activity and intraneuronal abeta in Alzheimer's disease.

beta-Amyloid peptide accumulation plays a central role in the pathogenesis of Alzheimer's disease. Aberrant beta-amyloid buildup in the brain has been shown to be present both in the extracellular space and within neurons. Synapses are important targets of beta-amyloid, and alterations in synapses better correlate with cognitive impairment than amyloid plaques or neurofibrillary tangles. The link between beta-amyloid and synapses became even tighter when it was discovered that beta-amyloid accumulates within synapses and that synaptic activity modulates beta-amyloid secretion. Currently, a central question in Alzheimer's disease research is what role synaptic activity plays in the disease process, and how specifically beta-amyloid is involved in the synaptic dysfunction that characterizes the disease.

Intraneuronal beta-amyloid accumulation and synapse pathology in Alzheimer's disease.

The aberrant accumulation of aggregated beta-amyloid peptides (Abeta) as plaques is a hallmark of Alzheimer's disease (AD) neuropathology and reduction of Abeta has become a leading direction of emerging experimental therapies for the disease. The mechanism(s) whereby Abeta is involved in the pathophysiology of the disease remain(s) poorly understood. Initially fibrils, and subsequently oligomers of extracellular Abeta have been viewed as the most important pathogenic form of Abeta in AD. More recently, the intraneuronal accumulation of Abeta has been described in the brain, although technical considerations and its relevance in AD have made this a controversial topic. Here, we review the emerging evidence linking intraneuronal Abeta accumulation to the development of synaptic pathology and plaques in AD, and discuss the implications of intraneuronal beta-amyloid for AD pathology, biology, diagnosis and therapy.

Synaptic activity reduces intraneuronal Abeta, promotes APP transport to synapses, and protects against Abeta-related synaptic alterations.

A central question in Alzheimer's disease research is what role synaptic activity plays in the disease process. Synaptic activity has been shown to induce beta-amyloid peptide release into the extracellular space, and extracellular beta-amyloid has been shown to be toxic to synapses. We now provide evidence that the well established synaptotoxicity of extracellular beta-amyloid requires gamma-secretase processing of amyloid precursor protein. Recent evidence supports an important role for intraneuronal beta-amyloid in the pathogenesis of Alzheimer's disease. We show that synaptic activity reduces intraneuronal beta-amyloid and protects against beta-amyloid-related synaptic alterations. We demonstrate that synaptic activity promotes the transport of the amyloid precursor protein to synapses using live cell imaging, and that the protease neprilysin is involved in reduction of intraneuronal beta-amyloid with synaptic activity.

Triterpenoid CDDO-methylamide improves memory and decreases amyloid plaques in a transgenic mouse model of Alzheimer's disease.

Oxidative stress is one of the earliest events in the pathogenesis of Alzheimer's disease (AD) and can markedly exacerbate amyloid pathology. Modulation of antioxidant and anti-inflammatory pathways represents an important approach for AD therapy. Synthetic triterpenoids have been found to facilitate antioxidant response and reduce inflammation in several models. We investigated the effect of the triterpenoid, 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic acid-MethylAmide (CDDO-MA) in Tg19959 mice, which carry the human amyloid precursor protein with two mutations. These mice develop memory impairments and amyloid plaques as early as 2-3 months of age. CDDO-MA was provided with chow (800 mg/kg) from 1 to 4 months of age. CDDO-MA significantly improved spatial memory retention and reduced plaque burden, Abeta42 levels, microgliosis, and oxidative stress in Tg19959 mice.

Internalized antibodies to the Abeta domain of APP reduce neuronal Abeta and protect against synaptic alterations.

Immunotherapy against beta-amyloid peptide (Abeta) is a leading therapeutic direction for Alzheimer disease (AD). Experimental studies in transgenic mouse models of AD have demonstrated that Abeta immunization reduces Abeta plaque pathology and improves cognitive function. However, the biological mechanisms by which Abeta antibodies reduce amyloid accumulation in the brain remain unclear. We provide evidence that treatment of AD mutant neuroblastoma cells or primary neurons with Abeta antibodies decreases levels of intracellular Abeta. Antibody-mediated reduction in cellular Abeta appears to require that the antibody binds to the extracellular Abeta domain of the amyloid precursor protein (APP) and be internalized. In addition, treatment with Abeta antibodies protects against synaptic alterations that occur in APP mutant neurons.