Injection Of Brain Extracts Research Articles

Learning from anti‐tau antibody clinical trials using computer simulation of spatial tau propagation

AbstractBackgroundBased on the tau spreading hypothesis, anti‐tau antibodies have been developed that can bind to and eliminate the misfolded tau protein during the short extracellular period. However, trials with tau antibodies have come up short in improving clinical outcome in Progressive Supranuclear Palsy (PSP) or Alzheimer’s Disease (AD). It is unclear whether the negative outcomes were due to insufficient target engagement and to what extent the change of tau CSF biomarkers reflected the brain pharmacodynamics.MethodA Quantitative Systems Pharmacology (QSP) computer platform of tau biology has been developed including tau species diversity and their respective concentration, neuronal activity‐dependent tau secretion, tau binding to membrane surface receptors HSPG and LRP1, tau‐antibody binding and subsequent internalization of tau at the synaptic cleft. This model is combined with a Physiology‐Based PharmacoKinetic (PBPK) model to derive brain target exposure of tau antibodies. In addition, a PBPK model is used to simulate the level of plasma and CSF biomarkers.ResultThe platform was calibrated with preclinical in‐vitro cellular assays and after injection of brain extract in Transgene mouse models and was further humanized using published data on seed‐competent tau from postmortem AD brain at different Braak stages. Combining the extracellular antibody exposure, the spatial dimensions and the dynamics of tau secretion, diffusion and binding to the membrane surface receptors, the model suggests that a very limited fraction (10‐20%) of misfolded tau protein can be captured by antibodies and cleared. Even less reduction (5‐10%) is predicted for Progressive Supranuclear Palsy due to the higher affinity of PSP‐4R tau for membrane surface receptors. The reductions in misfolded tau protein are greatest in earlier stages of the disease. With regard to epitope selection, we identify conditions where high specificity (i.e. monomeric vs misfolded tau) is superior to high sensitivity (affinity).ConclusionAdvanced Quantitative Systems Pharmacology computer modeling combining drug exposure dynamics with mechanistic modeling allows to (1) generate testable hypotheses for the outcomes of current therapeutic interventions, (2) support improved clinical trial designs in terms of dosing, patient selection, background amyloid therapy and disease stage and (3) ultimately prioritize targets for addressing tau pathology.

Intracellular Injection of Brain Extracts from Alzheimer's Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics.

Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimer's disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aβs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aβ oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract's ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aβ oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.

Supporting anti‐tau antibody trials using computer simulation of spatial tau propagation

AbstractBackgroundAlthough tau pathology is a major driver of AD pathology, the mere complexity of tau biology significantly hampers the identification and validation of the most impactful target. Recent clinical trials using antibodies against various forms of tau have come up short in improving cognitive readout.MethodA Quantitative Systems Pharmacology computer platform of tau biology has been developed including tau species diversity, neuronal activity‐dependent tau secretion, tau binding to membrane surface receptors, subsequent internalization at the synaptic cleft and along the axonal membrane following a diffusion gradient, slow motor‐mediated axonal transport, intracellular degradation and templating of monomeric tau by seed‐competent tau to form higher‐order aggregates. Polysynaptic progression is implemented by modeling secretion from the first synapse and uptake in the second axon.ResultThe platform was calibrated to reproduce preclinical experiments from in‐vitro cellular assays and after injection of brain extract in Tg models. The model is humanized using published data on seed‐competent tau from postmortem AD brain at different Braak stages and spatio‐temporal progression over neuronal projections. By exploring the effect of target mediated drug disposal of antibodies, we show that depending upon the disease state only between 15 and 30% of misfolded tau protein can be captured by antibodies currently in clinical trials. Introducing PSP specific uptake parameters from preclinical experiments, the simulations suggest an even lower efficacy due to the higher affinity of PSP‐tau for membrane surface receptors. We further identify conditions under which specificity (i.e. monomeric vs misfolded tau) of antibodies outweigths sensitivity (affinity). Finally, the simulations suggest that reduced oligomerization and increased degradation have a greater impact on the accumulation of seed‐competent tau in distal regions, likely because they act during the long intracellular residence of misfolded tau protein.ConclusionAdvanced Quantitative Systems Pharmacology computer modeling combining drug exposure with mechanistic modeling allows to ‘humanize’ preclinical experiments and aims to support tauopathy R&D projects.

Investigating astrocytes as mediators of tau spread.

Highly phosphorylated tau aggregates spread in a prion-like manner through tauopathy brain. Increasing evidence suggests that astrocytes may influence tau spread, with recent data describing mechanisms of tau uptake my astrocytes. However, the efficiency of uptake of different disease-associated tau species is not well defined, nor is the effects of tau uptake on astrocyte reactivity and function. We have investigated the relationship between tau seeds and reactive astrocytes in htau mice and in human astrocyte cultures. Tau was isolated from human postmortem control (CTRL), Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and Pick's disease (PiD) brain and injected intraperitoneally into 4-month old wild-type and htau mice. Brains and other tissues were collected for biochemical and immunohistochemical analysis of tau aggregated burden in different brain regions when mice reached 18 months of age. Human iPSC-derived astrocytes and iNPC-astrocytes were established and their gene and protein expression profiles characterised. Aggregated tau was isolated from postmortem human brain for spiking into astrocyte cultures. Cells were analysed for efficiency of tau uptake and astrocyte reactivity. Peripheral injection of brain extracts from AD, PSP and PiD brain resulted in regional differences in the patterns of tau aggregate accumulation in htau mouse brain. Tau pathology was commonly associated with reactive astrocytes. Cultures of human iPSC-derived astrocytes and iNPC-astrocytes that express mature astrocyte markers were established. Both types of cell culture were found to internalise tau extracts from human postmortem brain. Furthermore, preliminary analysis indicates alterations in astrocyte reactivity following tau uptake. Our data suggests that neurons in different regions are selectively vulnerable to tau aggregates from different tauopathies. Our human astrocyte cultures demonstrate that astrocytes can internalise tau seeds and alter their reactivity and potentially their functions. Further investigations will determine the mechanisms of tau uptake and the downstream effects on astrocyte signalling and functional changes, providing further insights into how astrocytes may affect the spread of tau and potentially contribute to selective regional vulnerability in tauopathies.

Investigating astrocytes as mediators of tau spread

AbstractBackgroundHighly phosphorylated tau aggregates are believed to spread in a prion‐like manner through tauopathy brain. We set out to determine the characteristics of tau deposition in mice expressing wild‐type human 3R and 4R tau, following peripheral injection of tau extracts from different tauopathies. Tau accumulations identified around astrocytes in the experimental mice prompted us to develop a human astrocyte culture model to further investigate tau uptake and spread.MethodTau was isolated from human postmortem control (CTRL), Alzheimer’s disease (AD), progressive supranuclear palsy (PSP) and Pick’s disease (PiD) brain. Extracts were injected intraperitoneally into 4‐month old wild‐type and htau mice, and at 18 month‐of‐age tissue was collected for biochemical and immunohistochemical assessment.Human iPSCs were directed into neural progenitor cells and terminally plated as astrocytes using a previously established protocol. Astrocytes were characterised using RT‐qPCR and immunocytochemical assessment. Cultures were spiked with the previously derived pathogenic tau isolated from human tissue. Cells were analysed immunocytochemically for tau uptake.ResultWe found that peripheral injection of brain extracts from AD, PSP and PiD brain results in regional differences in the patterns of tau aggregate accumulation in the mouse brain. Some tau pathology was associated with ‘activated’ astrocytes, similar to the pathology observed in the corresponding human postmortem brain. Cultures of human astrocytes that express mature cell markers were established.ConclusionOur data suggests that neurons in different regions are susceptible to different species of tau aggregates. It is possible that this might hint towards selective vulnerability in different tauopathies. The association of tau pathology with astrocytes in some cases suggests that astrocytes may play a role in tau spread. Our human astrocyte cultures will provide a model to determine how astrocytes may facilitate the spread of tau and potentially contribute to selective regional vulnerability in tauopathies.

Cell model for the identification and characterization of prion-like components from Alzheimer brain tissue

Intracerebral injection of brain extracts from Alzheimer's disease (AD) patients into appropriate mouse models was previously found to drastically accelerate the deposition of Aβ amyloid in the recipient animals indicating a prion-like activity. In this study we show that this prion-like activity can be also identified by using a cell culture model of Aβ plaque formation. Analysis of biochemical fractions of AD brain extract indicate that the seeding-activity correlated with the presence of Aβ peptide and Aβ-derived aggregates. In vitro-formed fibrils were also active but their activity was low and depending on the fibril structure and conditions of fibril formation. Our data indicate a conformational basis of the observed seeding effect and suggest the utility of our cell model for further studies on the prion-like activity of AD extracts.

Aβ seeds resist inactivation by formaldehyde.

Cerebral β-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated β-amyloid (Aβ) into young, pre-depositing Aβ precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated Aβ itself. Here we report that the β-amyloid-inducing activity of Alzheimer's disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated Aβ are maintained in fixed tissues. The resistance of Aβ seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of Aβ aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

Brain homogenates from human tauopathies induce tau inclusions in mouse brain

Filamentous inclusions made of hyperphosphorylated tau are characteristic of numerous human neurodegenerative diseases, including Alzheimer's disease, tangle-only dementia, Pick disease, argyrophilic grain disease (AGD), progressive supranuclear palsy, and corticobasal degeneration. In Alzheimer's disease and AGD, it has been shown that filamentous tau appears to spread in a stereotypic manner as the disease progresses. We previously demonstrated that the injection of brain extracts from human mutant P301S tau-expressing transgenic mice into the brains of mice transgenic for wild-type human tau (line ALZ17) resulted in the assembly of wild-type human tau into filaments and the spreading of tau inclusions from the injection sites to anatomically connected brain regions. Here we injected brain extracts from humans who had died with various tauopathies into the hippocampus and cerebral cortex of ALZ17 mice. Argyrophilic tau inclusions formed in all cases and following the injection of the corresponding brain extracts, we recapitulated the hallmark lesions of AGD, PSP and CBD. Similar inclusions also formed after intracerebral injection of brain homogenates from human tauopathies into nontransgenic mice. Moreover, the induced formation of tau aggregates could be propagated between mouse brains. These findings suggest that once tau aggregates have formed in discrete brain areas, they become self-propagating and spread in a prion-like manner.

Is it all about Contact? Neurodegeneration as a “Protein Freeze Tag Game” Inside the Central Nervous System

Is it all about Contact? Neurodegeneration as a “Protein Freeze Tag Game” Inside the Central Nervous System

Alzheimer brain-derived tau oligomers propagate pathology from endogenous tau

Intracerebral injection of brain extracts containing amyloid or tau aggregates in transgenic animals can induce cerebral amyloidosis and tau pathology. We extracted pure populations of tau oligomers directly from the cerebral cortex of Alzheimer disease (AD) brain. These oligomers are potent inhibitors of long term potentiation (LTP) in hippocampal brain slices and disrupt memory in wild type mice. We observed for the first time that these authentic brain-derived tau oligomers propagate abnormal tau conformation of endogenous murine tau after prolonged incubation. The conformation and hydrophobicity of tau oligomers play a critical role in the initiation and spread of tau pathology in the naïve host in a manner reminiscent of sporadic AD.

In Vivo Spreading of Tau Pathology

In Vivo Spreading of Tau Pathology

Transmission and spreading of tauopathy in transgenic mouse brain.

Hyperphosphorylated tau makes up the filamentous intracellular inclusions of several neurodegenerative diseases, including Alzheimer's disease 1. In the disease process neuronal tau inclusions first appear in transentorhinal cortex, from where they appear to spread to hippocampal formation and neocortex 2. Cognitive impairment becomes manifest when inclusions reach the hippocampus, with abundant neocortical tau inclusions and extracellular β-amyloid deposits being the defining pathological hallmarks of Alzheimer's disease. Abundant tau inclusions, in the absence of β-amyloid deposits, define Pick's disease, progressive supranuclear palsy, corticobasal degeneration and other diseases 1. Tau mutations cause familial forms of frontotemporal dementia, establishing that tau protein dysfunction is sufficient to cause neurodegeneration and dementia 3-5. Thus, transgenic mice expressing mutant (e.g. P301S) human tau in nerve cells exhibit the essential features of tauopathies, including neurodegeneration and abundant filaments made of hyperphosphorylated tau protein 6,7. In contrast, mouse lines expressing single isoforms of wild-type human tau do not produce tau filaments or display neurodegeneration 7,8. Here we have used tau-expressing lines to investigate whether experimental tauopathy can be transmitted. We show that the injection of brain extract from mutant P301S tau-expressing mice into the brain of transgenic wild-type tau-expressing animals induces the assembly of wild-type human tau into filaments and the spreading of pathology from the site of injection to neighbouring brain regions.

Regulation of sex pheromone production in the male Nauphoeta cinerea cockroach: Role of brain extracts, corpora allata??(CA), and juvenile hormone (JH)

The neuroendocrine mechanisms underlying pheromone regulation in cockroaches are unclear because of a lack of physiological and chemical data. The present report describes experiments designed to determine the role of the brain, corpora allata, and juvenile hormone III in the production of sex pheromone by male Nauphoeta cinerea cockroaches. The levels of two sex pheromone components, i.e., acetoin and 2-methylthiazolidine, were measured by gas chromatographic analysis of sternal gland extracts obtained from individual males. Allatectomy or decapitation performed up to 2 to 3 days after imaginal molt caused a decrease in sex phermone levels. Conversely decapitation or allatectomy performed after 3 to 4 days post-eclosion had almost no effect on sex pheromone levels. Injection of JHIII into allatectomized and decapitated males stimulated pheromone production while injection of brain extract had no effect. These results indicate that JHIII is involved in the differentiation of sternal glands and that regulation via pheromone biosynthesis activating neuropeptide (PBAN) does not occur in N. Cinerea cockroaches. Arch. Insect Biochem. Physiol. 40:165–172, 1999. © 1999 Wiley-Liss, Inc.

Juvenile hormone stimulates pheromonotropic brain factor release in the female black cutworm, Agrotis ipsilon

Sex pheromone biosynthesis of various insects has been shown to be controlled by a brain neuropeptide (PBAN) and in some cases by juvenile hormone (JH) from the corpora allata (CA). However, the mode of action of JH in the control of sex pheromone biosynthesis is still unclear. Although pheromonotropic activity was detected in their brains, females of the black cutworm, Agrotis ipsilon, lacking CA did not produce sex pheromone. Pheromone production could be restored in allatectomized female moths by injection of brain extracts or by injection of synthetic PBAN or JH-III. Injection of synthetic JH-III did not stimulate pheromone production in decapitated females. These results indicate that JH secreted by CA control sex pheromone biosynthesis of A. ipsilon females by stimulating pheromonotropic brain factor (PBAN-like factor) release from the central nervous system and not by controlling PBAN-like production or by priming PBAN-like target glandular cells.

Increase of paradoxical sleep (PS) by intraperitoneal injection of brain extract from PS-deprived rats.

Intraperitoneal injection of a brain extract obtained from paradoxical sleep-(PS) deprived donor rats resulted in a small but significant increase of PS in normal recipient rats. Brain extract of non-deprived control rats was without effect. The results provide further evidence for the existence of a PS-inducing factor accumulating in the brain during PS deprivation.

Postural changes in healthy rats after intracranial injection of brain extracts from animals with experimental vestibulopathy

Postural changes in healthy rats after intracranial injection of brain extracts from animals with experimental vestibulopathy

Neuroendocrine control of hydration in megascolecid earthworms

Abstract o 1. Brain removal frequently produced counterative effects on the rapid weight gain in worms of the genus Pheretima placed in deionized water. 2. The effects are prevented by injection of brain extract, but not by extracts of ventral nerve cord or body wall. 3. Thus the rapid weight gain caused by water uptake may be under neuroendocrine control of the brain. 4. Paraldehyde-fuchsin positive neurons in the brain temporarily decrease to 30% or less immediately after submersion of the animals in water. 5. It is thus suggested that the brain neurosecretory cells are possibly the source of a chemical factor which affects rapid weight gain.

Induction of postural asymmetry in an intact recipient by brain extract from a donor with the same syndrome

An attempt was made at the direct transfer of postural asymmetry, following removal of half the anterior lobe of the cerebellum in rats, to a recipient. Asymmetry of the hind limbs was shown to appear in spinal animals after injection of brain extract of pathological donors into their subdural space below the level of transection. The specificity of the resulting asymmetry depending on the side of injury to the donor's cerebellum was revealed. The asymmetry of the recipient completely copied the asymmetry of the donor. Brain extracts from control animals did not induce asymmetry. Additional confirmation was obtained of the polypeptide nature of the factor stimulating the development of postural asymmetry. Treatment of the extract with pronase deprived it of its activity.

Male accessory gland substance of Melanoplus sanguinipes: An oviposition stimulant under the control of the corpus allatum

Abstract The accessory glands of male Melanoplus sanguinipes contain an oviposition stimulant. Injection of gland extracts from mature males induces oviposition in 75 per cent of capable virgin females within 24 hr. Injection of gland extracts from allatectomized males produces no stimulatory effect. Gland extracts from mature males contain two antigens which cannot be detected in gland extracts from allatectomized males. However, both antigens can be detected in gland extracts from allatectomized males 3 days after treatment with juvenile hormone. Anion-exchange chromatography of mature gland extracts yielded two fractions which, when injected into virgin females, induced oviposition in 71 per cent of capable insects within 24 hr. These two fractions are immunologically identical to the two antigens which are absent from gland extracts of allatectomized males. We suggest that synthesis of the oviposition stimulant in Melanoplus is controlled by the corpus allatum. Injection of brain extract also induces oviposition in 100 per cent of capable virgin females within 24 hr. A possible role for the brain in the oviposition process is discussed.

Increased susceptibility to audiogenic seizure resulting from injection of brain extract from acoustically primed mice

Mice of the C57BL/6J strain, which are normally nonsusceptible to audiogenic seizures, can be made susceptible by prior intense auditory stimulation. Since this “behavioral pattern” fits a general definition of learning, it was thought that effects of acoustical priming might be transferable in a manner similar to the transfer of learned behavior via injection of trained brain extract. Naive mice were injected with extract taken from the brains of primed mice. Using littermate controls that consisted of both noninjected mice and mice injected with either saline or extract from nonprimed donors, it was concluded that injection of brain extract from acoustically primed mice increased the susceptibility to audiogenic seizure in C57BL/6J mice.