Soluble Brain Research Articles

Plasma Exchange Reduces Aβ Levels in Plasma and Decreases Amyloid Plaques in the Brain in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common type of dementia, characterized by the abnormal accumulation of protein aggregates in the brain, known as neurofibrillary tangles and amyloid-β (Aβ) plaques. It is believed that an imbalance between cerebral and peripheral pools of Aβ may play a relevant role in the deposition of Aβ aggregates. Therefore, in this study, we aimed to evaluate the effect of the removal of Aβ from blood plasma on the accumulation of amyloid plaques in the brain. We performed monthly plasma exchange with a 5% mouse albumin solution in the APP/PS1 mouse model from 3 to 7 months old. At the endpoint, total Aβ levels were measured in the plasma, and soluble and insoluble brain fractions were analyzed using ELISA. Brains were also analyzed histologically for amyloid plaque burden, plaque size distributions, and gliosis. Our results showed a reduction in the levels of Aβ in the plasma and insoluble brain fractions. Interestingly, histological analysis showed a reduction in thioflavin-S (ThS) and amyloid immunoreactivity in the cortex and hippocampus, accompanied by a change in the size distribution of amyloid plaques, and a reduction in Iba1-positive cells. Our results provide preclinical evidence supporting the relevance of targeting Aβ in the periphery and reinforcing the potential use of plasma exchange as an alternative non-pharmacological strategy for slowing down AD pathogenesis.

Specific post-translational modifications of soluble tau protein distinguishes Alzheimer’s disease and primary tauopathies

Tau protein aggregates in several neurodegenerative disorders, referred to as tauopathies. The tau isoforms observed in post mortem human brain aggregates is used to classify tauopathies. However, distinguishing tauopathies ante mortem remains challenging, potentially due to differences between insoluble tau in aggregates and soluble tau in body fluids. Here, we demonstrated that tau isoforms differ between tauopathies in insoluble aggregates, but not in soluble brain extracts. We therefore characterized post-translational modifications of both the aggregated and the soluble tau protein obtained from post mortem human brain tissue of patients with Alzheimer’s disease, cortico-basal degeneration, Pick’s disease, and frontotemporal lobe degeneration. We found specific soluble signatures for each tauopathy and its specific aggregated tau isoforms: including ubiquitination on Lysine 369 for cortico-basal degeneration and acetylation on Lysine 311 for Pick’s disease. These findings provide potential targets for future development of fluid-based biomarker assays able to distinguish tauopathies in vivo.

An oligomer‐directed Aβ antibody decreases synaptotoxic oligomeric Aβ in brain and improves behavioral abnormality in humanized APP knock‐in mice

AbstractBackgroundThere is strong evidence that soluble oligomers of amyloid beta protein (oAβ) help initiate the pathogenic cascade of Alzheimer’s disease (AD), which suggests therapeutic strategies targeting oAβ over monomeric or fibrillar Aβ. A new antibody, 71A1, is 1∼00‐fold more sensitive for oAβ than synthetic monomers. The material that 71A1 specifically immunoprecipitates from AD soluble brain extracts impairs synaptic function as much as does the full extract. In accord, pre‐incubating brain extracts with 71A1 neutralizes its synaptotoxicity. 71A1 has potentially unique activities against disease‐relevant oAβ, making it a novel candidate for treating AD.Methods15 mg/kg 71A1 or anti‐KLH (negative control) was administered i.p. to 45 humanized APP knock‐in mice (APPNLGF/NLGF) weekly from 8 to 20 weeks of age. Cognition was assessed by spontaneous alternation (Y‐maze). Brains were harvested at age 21‐wk for biochemical, electrophysiological and immunohistochemical analyses. Diffusible (“soaking”) extracts of the brains were prepared to measure monomeric and oligomeric Aβ using homebrew ultra‐sensitive assays. Aliquots of the same soaking extracts were used to treat wild‐type mouse hippocampal slices and measure long‐term potentiation (LTP) to assess synaptotoxicity in the APPNLGF/NLGF brains post‐treatment.Results71A1 recognized oAβ and neutralized its synaptotoxicity in APPNLGF/NLGF mouse brain extracts. 71A1 effectively treated male APPNLGF/NLGF mice, which improved cognition significantly. The antibody reduced brain Aβ (1‐42) and oAβ levels with a trend toward statistical significance. Mouse brains with less oAβ had less synaptotoxicity as assessed by LTP.ConclusionsThese results demonstrate 71A1 is an oligomer‐preferring monoclonal antibody as a therapeutic candidate for AD, decreasing brain oAβ and oAβ‐induced synaptotoxicity. We believe this provides the first proof‐of‐concept mouse trial using an antibody that has been carefully characterized as oAβ‐preferring. We observed 1) a correlation between oAβ concentration and its synaptotoxicity; and 2) that 71A1 ameliorated aspects of cognitive impairment in APPNLGF/NLGF mice by targeting oAβ.

Visualization of tau oligomers in TauP301L‐BiFC transgenic mice exhibits the progression of tauopathy

AbstractBackgroundAccumulation of abnormal tau aggregates in the brain is a pathological hallmark of multiple neurodegenerative disorders including Alzheimer’s disease (AD). Increasing evidence suggests that soluble tau aggregates play a key role in tau pathology as neurotoxic species causing neuronal cell death and act as prion‐like seeds mediating tau propagation. Despite the pathological relevance, there is a paucity of methods to monitor tau oligomerization in the brain. Therefore, we have developed a novel tau transgenic mouse model as a tool to monitor tau self‐assembly in the brain.MethodWe introduced bimolecular fluorescence complementation (BiFC) technique to human tau containing a P301L mutation to generate a novel tau transgenic mouse, named TauP301L‐BiFC. Tau self‐assembly, represented by BiFC fluorescence was monitored and quantified in the brain of transgenic TauP301L‐BiFC mice. Behavior and biochemical differences were assessed between 3 and 12 months for the characterization of TauP301L‐BiFC mice. In vivo efficacy of methylene blue, a tau aggregation inhibitor, was evaluated for further validation of TauP301L‐BiFC mice as an in vivo model of tauopathy.ResultsTauP301L‐BiFC mice showed soluble tau oligomerization from 3 months, showing significantly enriched BiFC fluorescence in the brain. Then, massive tau fragmentation occurred at 6 months showing dramatically decreased TauP301L‐BiFC fluorescence. The fragmented tau species served as a seed for insoluble tau aggregation. In a result, insoluble TauP301L‐BiFC aggregates co‐aggregated with endogenous mouse tau accumulated in the brain, showing subsequently increased BiFC fluorescence from 9 months. Neuronal degeneration and cognitive deficits were observed from 12 months of age. Administration of methylene blue significantly reduced tau‐BiFC fluorescence in the brain of TauP301L‐BiFC mice, presenting a significant decrease in the level of matured tau‐BiFC complexes in the soluble brain lysates. TauP301L‐BiFC mouse model demonstrated that methylene blue reduced the amount of soluble tau oligomers in the brain, resulting in the prevention of cognitive impairments.ConclusionThe tau oligomerization was able to monitor and quantify in the brain of TauP301L‐BiFC mice. Thus, we assure that TauP301L‐BiFC mice are a bonafide animal tool to monitor pathological tau oligomerization in AD and other tauopathies.

APOE4 exacerbates α-synuclein seeding activity and contributes to neurotoxicity in Alzheimer’s disease with Lewy body pathology

Approximately half of Alzheimer’s disease (AD) brains have concomitant Lewy pathology at autopsy, suggesting that α-synuclein (α-SYN) aggregation is a regulated event in the pathogenesis of AD. Genome-wide association studies revealed that the ε4 allele of the apolipoprotein E (APOE4) gene, the strongest genetic risk factor for AD, is also the most replicated genetic risk factor for Lewy body dementia (LBD), signifying an important role of APOE4 in both amyloid-β (Aβ) and α-SYN pathogenesis. How APOE4 modulates α-SYN aggregation in AD is unclear. In this study, we aimed to determine how α-SYN is associated with AD-related pathology and how APOE4 impacts α-SYN seeding and toxicity. We measured α-SYN levels and their association with other established AD-related markers in brain samples from autopsy-confirmed AD patients (N = 469), where 54% had concomitant LB pathology (AD + LB). We found significant correlations between the levels of α-SYN and those of Aβ40, Aβ42, tau and APOE, particularly in insoluble fractions of AD + LB. Using a real-time quaking-induced conversion (RT-QuIC) assay, we measured the seeding activity of soluble α-SYN and found that α-SYN seeding was exacerbated by APOE4 in the AD cohort, as well as a small cohort of autopsy-confirmed LBD brains with minimal Alzheimer type pathology. We further fractionated the soluble AD brain lysates by size exclusion chromatography (SEC) ran on fast protein liquid chromatography (FPLC) and identified the α-SYN species (~ 96 kDa) that showed the strongest seeding activity. Finally, using human induced pluripotent stem cell (iPSC)-derived neurons, we showed that amplified α-SYN aggregates from AD + LB brain of patients with APOE4 were highly toxic to neurons, whereas the same amount of α-SYN monomer was not toxic. Our findings suggest that the presence of LB pathology correlates with AD-related pathologies and that APOE4 exacerbates α-SYN seeding activity and neurotoxicity, providing mechanistic insight into how APOE4 affects α-SYN pathogenesis in AD.

Absence of tissue transglutaminase reduces amyloid-beta pathology in APP23 mice.

AimsAlzheimer's disease (AD) is characterised by amyloid‐beta (Aβ) aggregates in the brain. Targeting Aβ aggregates is a major approach for AD therapies, although attempts have had little to no success so far. A novel treatment option is to focus on blocking the actual formation of Aβ multimers. The enzyme tissue transglutaminase (TG2) is abundantly expressed in the human brain and plays a key role in post‐translational modifications in Aβ resulting in covalently cross‐linked, stable and neurotoxic Aβ oligomers.In vivo absence of TG2 in the APP23 mouse model may provide evidence that TG2 plays a key role in development and/or progression of Aβ‐related pathology.MethodsHere, we compared the effects on Aβ pathology in the presence or absence of TG2 using 12‐month‐old wild type, APP23 and a crossbreed of the TG2−/− mouse model and APP23 mice (APP23/TG2−/−).ResultsUsing immunohistochemistry, we found that the number of Aβ deposits was significantly reduced in the absence of TG2 compared with age‐matched APP23 mice. To pinpoint possible TG2‐associated mechanisms involved in this observation, we analysed soluble brain Aβ1–40, Aβ1–42 and/or Aβ40/42 ratio, and mRNA levels of human APP and TG2 family members present in brain of the various mouse models. In addition, using immunohistochemistry, both beta‐pleated sheet formation in Aβ deposits and the presence of reactive astrocytes associated with Aβ deposits were analysed.ConclusionsWe found that absence of TG2 reduces the formation of Aβ pathology in the APP23 mouse model, suggesting that TG2 may be a suitable therapeutic target for reducing Aβ deposition in AD.

The Transglutaminase-2 Interactome in the APP23 Mouse Model of Alzheimer's Disease.

Amyloid-beta (Aβ) deposition in the brain is closely linked with the development of Alzheimer’s disease (AD). Unfortunately, therapies specifically targeting Aβ deposition have failed to reach their primary clinical endpoints, emphasizing the need to broaden the search strategy for alternative targets/mechanisms. Transglutaminase-2 (TG2) catalyzes post-translational modifications, is present in AD lesions and interacts with AD-associated proteins. However, an unbiased overview of TG2 interactors is lacking in both control and AD brain. Here we aimed to identify these interactors using a crossbreed of the AD-mimicking APP23 mouse model with wild type and TG2 knock-out (TG2−/−) mice. We found that absence of TG2 had no (statistically) significant effect on Aβ pathology, soluble brain levels of Aβ1–40 and Aβ1–42, and mRNA levels of TG family members compared to APP23 mice at 18 months of age. Quantitative proteomics and network analysis revealed a large cluster of TG2 interactors involved in synaptic transmission/assembly and cell adhesion in the APP23 brain typical of AD. Comparative proteomics of wild type and TG2−/− brains revealed a TG2-linked pathological proteome consistent with alterations in both pathways. Our data show that TG2 deletion leads to considerable network alterations consistent with a TG2 role in (dys)regulation of synaptic transmission and cell adhesion in APP23 brains.

Soluble brain homogenates from diverse human and mouse sources preferentially seed diffuse Aβ plaque pathology when injected into newborn mouse hosts.

Seeding of pathology related to Alzheimer's disease (AD) and Lewy body disease (LBD) by tissue homogenates or purified protein aggregates in various model systems has revealed prion-like properties of these disorders. Typically, these homogenates are injected into adult mice stereotaxically. Injection of brain lysates into newborn mice represents an alternative approach of delivering seeds that could direct the evolution of amyloid-β (Aβ) pathology co-mixed with either tau or α-synuclein (αSyn) pathology in susceptible mouse models. Homogenates of human pre-frontal cortex were injected into the lateral ventricles of newborn (P0) mice expressing a mutant humanized amyloid precursor protein (APP), human P301L tau, human wild type αSyn, or combinations thereof. The homogenates were prepared from AD and AD/LBD cases displaying variable degrees of Aβ pathology and co-existing tau and αSyn deposits. Behavioral assessments of APP transgenic mice injected with AD brain lysates were conducted. For comparison, homogenates of aged APP transgenic mice that preferentially exhibit diffuse or cored deposits were similarly injected into the brains of newborn APP mice. We observed that lysates from the brains with AD (Aβ+, tau+), AD/LBD (Aβ+, tau+, αSyn+), or Pathological Aging (Aβ+, tau-, αSyn-) efficiently seeded diffuse Aβ deposits. Moderate seeding of cerebral amyloid angiopathy (CAA) was also observed. No animal of any genotype developed discernable tau or αSyn pathology. Performance in fear-conditioning cognitive tasks was not significantly altered in APP transgenic animals injected with AD brain lysates compared to nontransgenic controls. Homogenates prepared from aged APP transgenic mice with diffuse Aβ deposits induced similar deposits in APP host mice; whereas homogenates from APP mice with cored deposits induced similar cored deposits, albeit at a lower level. These findings are consistent with the idea that diffuse Aβ pathology, which is a common feature of human AD, AD/LBD, and PA brains, may arise from a distinct strain of misfolded Aβ that is highly transmissible to newborn transgenic APP mice. Seeding of tau or αSyn comorbidities was inefficient in the models we used, indicating that additional methodological refinement will be needed to efficiently seed AD or AD/LBD mixed pathologies by injecting newborn mice.

Cerebral Phospho-Tau Acts Synergistically with Soluble Aβ42 Leading to Mild Cognitive Impairment in AAV-AD Rats.

Alzheimer's disease (AD) is a continuum of events beginning with an increase in brain soluble Aβ42 followed by the appearance of hyperphosphorylated tau (P-tau, asymptomatic stage). Mild Cognitive Impairment (MCI) then appears (prodromal stage). However, the individual contribution of these two soluble proteins in the onset of the first cognitive symptoms remains unclear. We sought to understand the specific impact of p-tau on the development of MCI in the AAV-AD rat model, a model of late-onset Alzheimer's disease (LOAD) predementia. We specifically reduced the phosphorylation level of tau while leaving Aβ42 levels unchanged using a DYRK1A protein kinase inhibitor, Leucettine L41, in an adeno-associated virus-based Alzheimer's disease (AAV-AD) rat model. Leucettine L41 was administered by intraperitoneal injection at 20 mg/kg per day in AAV-AD rats from 9 (late asymptomatic phase) to 10 (prodromal phase) months of age. Decreased soluble forms of P-tau induced by chronic administration of Leucettine L41 did not change soluble Aβ42 levels but prevented MCI onset in 10-month-old AAV-AD rats. The present study argues that P-tau is required to induce the development of MCI. Consistent with our previous findings that soluble Aβ42 is also required for MCI onset, the data obtained in the AAV-AD rat model confirm that the transition from the asymptomatic to the prodromal stage may be caused by the combined presence of both soluble brain forms of Aβ42 and p-tau, suggesting that the development of MCI may be the consequence of their synergistic action.

CSF Aβ42 and Aβ40 and their relation to brain soluble and insoluble Aβ in the 5xFAD mouse model of Alzheimer's disease.

In patients with AD, CSF Aβ42 is reduced while Aβ40 remains unchanged. It has been suggested that altered CSF Aβ42 is due to aggregation of this peptide into insoluble plaques, resulting in less soluble Aβ42 available for secretion to the CSF. However, the relations between soluble and insoluble Aβ42 and Aβ40 in the brain and the concentrations of these Aβ peptides in CSF are not well studied. CSF and cortical brain tissue was collected from 2, 4, 6, and 12 months old male and female 5xFAD mice (n=45). CSF Aβ42 and Aβ40 concentrations were measured using Single molecule array (Simoa) technology. Brain sections were prepared and immunohistochemically (IHC) stained using antibodies specific for Aβ42 and Aβ40. The concentrations of Aβ42 and Aβ40 in soluble (extracted with TBS) and insoluble (extracted with formic acid) cortical brain fractions were determined by the Meso Scale Discovery technique. CSF Aβ42 was decreased over time whereas CSF Aβ40 remained unaltered (Fig 1). In the same mice, IHC revealed an age-related increased deposition of both Aβ42 and Aβ40 in insoluble plaques from 2 months of age (Fig 2). Moreover, measurements of Aβ42 and Aβ40 in soluble and insoluble cortical brain fractions showed increased concentrations of both peptides over time (Fig 3). CSF Aβ42 correlated inversely with cortical deposition of Aβ42 determined with IHC and the concentrations of Aβ42 in soluble and insoluble brain fractions. In contrast, no such correlations were found for Aβ40 (Fig 4). Although cortical levels of the two Aβ peptides were higher in females than in males, these sex differences were not reflected in CSF (Fig 5). Although significant depositions of both Aβ42 and Aβ40 were found in the brain, only Aβ42 was altered in CSF. Together with the finding that Aβ42 was increased, and not reduced, in soluble cortical brain fractions, this may suggest that mechanisms other than aggregation of Aβ42 into insoluble plaques contribute to decreased CSF concentrations of this Aβ peptide in 5xFAD mice. However, additional characterization of Aβ in the soluble brain fraction is needed to further understand its relation to the concentrations in CSF.

Non-denaturing affinity purification of soluble oligomeric Aβ from human brain using a novel calcium-sensitive monoclonal antibody.

The soluble fraction of human AD brain extracts contains the most bioactive oligomeric forms of Aβ (oAβ), which are low in abundance. Few methods exist to enrich for oAβ from soluble brain extracts without denaturing them. Purification of natural, human oAβ would allow further structural and biochemical study of this truly disease-relevant species. A panel of anti-oAβ monoclonal antibodies was developed by immunizing Trianni mice with synthetic Aβ aggregates. Clones were screened for oAβ selectivity and protection against human oAβ-induced injury in an in vitro neuritic integrity assay and in mouse hippocampal slice LTP recordings. Four antibodies with selectivity for oAβ over monomeric Aβ and protective effects were chosen for further study. Quantitative immunoprecipitations (IP followed by guanidine denaturation and monomer-specific ELISA) were performed on soluble extracts obtained by soaking cortical fragments bits in TBS to enrich for bioactive diffusible oAβ species. One antibody, B24, could IP oAβ only from non-dialyzed extracts. Iterative IPs were used to identify calcium as the soluble, dialyzable factor required for B24 binding to oAβ. Differential Scanning Fluorimetry (nano-DSF), Octet biolayer interferometry (BLI), and immunohistochemistry were further used to characterize B24 calcium-dependence and reversibility of binding to oAβ. B24 showed complete dependence on calcium at low millimolar levels for binding to soluble human oAβ, plaques, and synthetic Aβ protofibrils. Calcium induced a conformational change in B24. Binding of human soluble brain extracts to B24, washing, and elution with EGTA resulted in enrichment of oAβ. The recovered oAβ accounted for 20-40% of the initial input oAβ, accompanied by >400-fold reduction in total protein content. The recovered oAβ required guanidine hydrochloride for denaturation and detection by Aβ monomer-specific ELISA, implying that the recovered oAβ had not been denatured during purification. Calcium did not affect the size distribution of oAβ from human brain extracts by size exclusion chromatography. B24 is protective against natural oAβ-induced neuronal injury and binds oAβ in a calcium-dependent manner. This unique characteristic allows affinity purification of bioactive oAβ from human brain extracts for further study and drug development.

Unbiased high-content screening reveals Aβ- and tau-independent synaptotoxic activities in human brain homogenates from Alzheimer's patients and high-pathology controls.

Alzheimer’s disease (AD) is tightly correlated with synapse loss in vulnerable brain regions. It is assumed that specific molecular entities such as Aβ and tau cause synapse loss in AD, yet unbiased screens for synaptotoxic activities have not been performed. Here, we performed size exclusion chromatography on soluble human brain homogenates from AD cases, high pathology non-demented controls, and low pathology age-matched controls using our novel high content primary cultured neuron-based screening assay. Both presynaptic and postsynaptic toxicities were elevated in homogenates from AD cases and high pathology non-demented controls to a similar extent, with more modest synaptotoxic activities in homogenates from low pathology normal controls. Surprisingly, synaptotoxic activities were found in size fractions peaking between the 17–44 kDa size standards that did not match well with Aβ and tau immunoreactive species in these homogenates. The fractions containing previously identified high molecular weight soluble amyloid beta aggregates/”oligomers” were non-toxic in this assay. Furthermore, immunodepletion of Aβ and tau did not reduce synaptotoxic activity. This result contrasts with previous findings involving the same methods applied to 3xTg-AD mouse brain extracts. The nature of the synaptotoxic species has not been identified. Overall, our data indicates one or more potential Aβ and tau independent synaptotoxic activities in human AD brain homogenates. This result aligns well with the key role of synaptic loss in the early cognitive decline and may provide new insight into AD pathophysiology.

Isoform-specific dysregulation of AMP-activated protein kinase signaling in a non-human primate model of Alzheimer's disease

AMP-activated protein kinase (AMPK) is a molecular sensor that is critical for the maintenance of cellular energy homeostasis, disruption of which has been indicated in multiple neurodegenerative diseases including Alzheimer's disease (AD). Mammalian AMPK is a heterotrimeric complex and its enzymatic α subunit exists in two isoforms: AMPKα1 and AMPKα2. Here we took advantage of a recently characterized non-human primate (NHP) model with sporadic AD-like neuropathology to explore potential relationships between AMPK signaling and AD-like neuropathology. Subjects were nine female vervet monkeys aged 19.5 to 23.4 years old. Subjects were classified into three groups, control lacking AD pathology (n = 3), moderate AD pathology (n = 3), and more severe AD Pathology (n = 3). We found increased activity (assessed by phosphorylation) of AMPKα2 in hippocampi of NHP with AD-like neuropathology, compared to the subjects without AD pathology, with no alterations of AMPKα1 activity. Across all subjects, CSF Abeta42 was inversely associated with cerebral amyloid plaque density. Further, Aβ plaque burden is correlated with levels of either soluble or insoluble brain Aβ measurement. Unbiased mass spectrometry based proteomics studies combined with bioinformatics analysis revealed that many of the dysregulated proteins characteristic of AD neuropathology are associated with AMPK signaling. Our findings on the AMPK molecular signaling cascades provide further support for use of the NHP model to investigate new therapeutic strategies and development of novel biomarkers for Alzheimer's disease.

Neuron-specific enolase in cerebrospinal fluid as a biomarker of brain damage in infants with hypoxic-ischemic encephalopathy.

Neuron-specific enolase in cerebrospinal fluid as a biomarker of brain damage in infants with hypoxic-ischemic encephalopathy.

SETD7-mediated monomethylation is enriched on soluble Tau in Alzheimer\u2019s disease

BackgroundHuman tauopathies including Alzheimer’s disease (AD) are characterized by alterations in the post-translational modification (PTM) pattern of Tau, which parallel the formation of insoluble Tau aggregates, neuronal dysfunction and degeneration. While PTMs on aggregated Tau have been studied in detail, much less is known about the modification patterns of soluble Tau. Furthermore, PTMs other than phosphorylation have only come into focus recently and are still understudied. Soluble Tau species are likely responsible for the spreading of pathology during disease progression and are currently being investigated as targets for immunotherapies. A better understanding of their biochemical properties is thus of high importance.MethodsWe used a mass spectrometry approach to characterize Tau PTMs on a detergent-soluble fraction of human AD and control brain tissue, which led to the discovery of novel lysine methylation events. We developed specific antibodies against Tau methylated at these sites and biochemically characterized methylated Tau species in extracts from human brain, the rTg4510 mouse model and in hiPSC-derived neurons.ResultsOur study demonstrates that methylated Tau levels increase with Tau pathology stage in human AD samples as well as in a mouse model of Tauopathy. Methylated Tau is enriched in soluble brain extracts and is not associated with hyperphosphorylated, high molecular weight Tau species. We also show that in hiPSC-derived neurons and mouse brain, methylated Tau preferentially localizes to the cell soma and nuclear fractions and is absent from neurites. Knock down and inhibitor studies supported by proteomics data led to the identification of SETD7 as a novel lysine methyltransferase for Tau. SETD7 specifically methylates Tau at K132, an event that facilitates subsequent methylation at K130.ConclusionsOur findings indicate that methylated Tau has a specific somatic and nuclear localization, suggesting that the methylation of soluble Tau species may provide a signal for their translocation to different subcellular compartments. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may shed light onto this disease-associated phenomenon.

Tau Is Truncated in Five Regions of the Normal Adult Human Brain.

The truncation of Tau is thought to be important in promoting aggregation, with this feature characterising the pathology of dementias such as Alzheimer disease. Antibodies to the C-terminal and N-terminal regions of Tau were employed to examine Tau cleavage in five human brain regions: the entorhinal cortex, prefrontal cortex, motor cortex, hippocampus, and cerebellum. These were obtained from normal subjects ranging in age from 18 to 104 years. Tau fragments of approximately 40 kDa and 45 kDa with an intact N-terminus retained were found in soluble and insoluble brain fractions. In addition, smaller C-terminal Tau fragments ranging in mass from 17 kDa to 25 kDa were also detected. These findings are consistent with significant Tau cleavage taking place in brain regions from 18 years onwards. It appears that site-specific cleavage of Tau is widespread in the normal human brain, and that large Tau fragments that contain the N-terminus, as well as shorter C-terminal Tau fragments, are present in brain cells across the age range.

Transcriptomic correlates of neurite degeneration due to human brain‐derived Aβ and protection by clinical anti‐Aβ antibodies

AbstractBackgroundThe proximate cause of dementia in Alzheimer disease (AD) is the dystrophy and loss of synapses and neurites, and the eventual death of neurons. Upstream events include hyperphosphorylation and aggregation of tau, and further upstream is the formation of soluble oligomers of Aβ. The molecular details which link these three and other events within the “amyloid cascade” remain murky, but elucidating them will likely reveal new therapeutic targets.MethodTo dissect early steps in the Aβ oligomer‐driven neuritotoxicity, we recently developed an in vitro system in which human brain‐derived soluble Aβ is applied to iPSC‐derived human neurons (iNs) to induce neurite degeneration (Jin et al, Nat Commun. 2018; 9(1):2676). Soluble extracts from AD brain were either immunodepleted of Aβ with a polyclonal antiserum or mock‐immunodepleted with rabbit IgG, then applied at 21 DIV to iPSC‐derived neurons that had been induced by Neurogenin‐2 expression. Neurite integrity was tracked using the NeuroTrack module of the IncuCyte ZOOM system for live‐cell analysis. Various humanized anti‐Aβ antibodies were added at concentrations from 0.5 to 3 ug/mL along with the control antibody bevacizumab (Avastin).ResultImmunodepletion with the polyclonal antibody resulted in efficient removal of Aβ. Mock‐immunodepleted, but not immunodepleted, human soluble brain extracts caused quantifiable neuritotoxicity using this unbiased, automated imaging system. These effects were rescued to different degrees by different anti‐Aβ antibodies but not by Avastin.ConclusionHuman AD brain‐derived soluble extracts cause neuritotoxicity to human neurons in an Aβ oligomer‐dependent manner. This effect can be decreased by anti‐Aβ antibodies currently in clinical trials, including aducanumab and BAN2401. We are now performing unbiased transcriptomic and proteomic profiling of the iNs after treatment with human‐derived soluble Aβ oligomers to determine the immediate molecular events that precede and accompany Aβ‐dependent neuritotoxicity. These results will be compared with recently‐published single‐cell transcriptomic data from AD brain to identify potential therapeutic targets.

Correction of microtubule defects within Aβ plaque-associated dystrophic axons results in lowered Aβ release and plaque deposition.

The hallmark pathologies of the Alzheimer's disease (AD) brain are amyloid beta (Aβ)-containing senile plaques and neurofibrillary tangles formed from the microtubule (MT)-binding tau protein. Tau becomes hyperphosphorylated and disengages from MTs in AD, with evidence of resulting MT structure/function defects. Brain-penetrant MT-stabilizing compounds can normalize MTs and axonal transport in mouse models with tau pathology, thereby reducing neuron loss and decreasing tau pathology. MT dysfunction is also observed in dystrophic axons adjacent to Aβ plaques, resulting in accumulation of amyloid precursor protein (APP) and BACE1 with the potential for enhanced localized Aβ generation. We have examined whether the brain-penetrant MT-stabilizing compound CNDR-51657 might decrease plaque-associated axonal dystrophy and Aβ release in 5XFAD mice that develop an abundance of Aβ plaques. Administration of CNDR-51657 to 1.5-month-old male and female 5XFAD mice for 4 or 7 weeks led to decreased soluble brain Aβ that coincided with reduced APP and BACE1 levels, resulting in decreased formation of insoluble Aβ deposits. These data suggest a vicious cycle whereby initial Aβ plaque formation causes MT disruption in nearby axons, resulting in the local accumulation of APP and BACE1 that facilitates additional Aβ generation and plaque deposition. The ability of a MT-stabilizing compound to attenuate this cycle, and also reduce deficits resulting from reduced tau binding to MTs, suggests that molecules of this type hold promise as potential AD therapeutics.

Constant Levels of Tau Phosphorylation in the Brain of htau Mice

Excessive tau phosphorylation is the hallmark of tauopathies. Today’s research thus focusses on the development of drugs targeting this pathological feature. To test new drugs in preclinical studies, animal models are needed that properly mimic this pathological hallmark. The htau mouse is a well-known model expressing human but lacking murine tau, allowing to evaluate the efficacy of tau modifying compounds without interference from murine tau. Htau mice are well-characterized for tau pathology at older age, although it is often not specified on which genetic background analyzed animals were bred. Since it was shown that the genetic background can influence the pathology, we evaluated the phosphorylation status of young and adult htau mice on a C57BL/6J background by analyzing ptau Ser202 and ptau Ser396 levels in the cortex and hippocampus of 3 and 12 month old animals by immunofluorescent labelling. Additionally, we evaluated total tau, ptau Thr231 and ptau Thr181 in the soluble and insoluble brain fraction of 3–15 month old htau mice by immunosorbent assay. Our results show that ptau levels of all analyzed residues and age groups are similar without strong increases over age. These data show that tau is already phosphorylated at the age of 3 months suggesting that phosphorylation starts even earlier. The early start of tau phosphorylation in htau mice enables the use of these mice for efficacy studies already at very young age.

Animal model for sporadic dementia of Alzheimer’s type (SDAT) using streptozotocin and lipopolysaccharide combinations in rats

Sporadic dementia of Alzheimer’s type (SDAT) pathogenesis has not been revealed completely due to the difficulty in creating an appropriate animal model. The purpose of this study was to investigate the effect of single-dose intraperitoneal (IP) induction of streptozotocin (STZ) and lipopolysaccharide (LPS) on the β-amyloid levels and the brain function of experimental rats. Eighteen rats were divided into three groups i.e. control, TRE1 (STZ 60 mg.kg-1 BW + LPS 3 mg.kg-1 BW), and TRE2 (STZ 30 mg.kg-1 BW + LPS 1.5 mg.kg-1 BW). The substances were administered in a single dose. Behavioral tests were started at day-30 after injection, we performed Morris water maze (MWM) and novel object recognition (NOR) tests. Afterward, we measured whole brain and serum β-amyloid levels, as one of the biomarkers of Alzheimer’s Disease (AD), using the ELISA method. In MWM tests, the escape latency and time spent in the target quadrant of treatment groups were significantly higher than those in control at the day-5 MWM test and probe trial. The rats in treatment groups have negative discrimination indexes in NOR tasks, indicating that the rats could not remember the familiar object. Intraperitoneal STZ and LPS significantly increase soluble brain β-amyloid levels of treatment groups than those in the control group. In conclusion, the treatment of STZ (60 mg.kg-1 BW) and LPS (3 mg.kg-1 BW) indicated spatial and recognition memory impairment, along with an increase of brain soluble β-amyloid level in rats.