Tau Cleavage Contributes to Cognitive Dysfunction in Strepto-Zotocin-Induced Sporadic Alzheimer’s Disease (sAD) Mouse Model

Tau cleavage plays a crucial role in the onset and progression of Alzheimer’s Disease (AD), a widespread neurodegenerative disease whose incidence is expected to increase in the next years. While genetic and familial forms of AD (fAD) occurring early in life represent less than 1%, the sporadic and late-onset ones (sAD) are the most common, with ageing being an important risk factor. Intracerebroventricular (ICV) infusion of streptozotocin (STZ)—a compound used in the systemic induction of diabetes due to its ability to damage the pancreatic β cells and to induce insulin resistance—mimics in rodents several behavioral, molecular and histopathological hallmarks of sAD, including memory/learning disturbance, amyloid-β (Aβ) accumulation, tau hyperphosphorylation, oxidative stress and brain glucose hypometabolism. We have demonstrated that pathological truncation of tau at its N-terminal domain occurs into hippocampi from two well-established transgenic lines of fAD animal models, such as Tg2576 and 3xTg mice, and that it’s in vivo neutralization via intravenous (i.v.) administration of the cleavage-specific anti-tau 12A12 monoclonal antibody (mAb) is strongly neuroprotective. Here, we report the therapeutic efficacy of 12A12mAb in STZ-infused mice after 14 days (short-term immunization, STIR) and 21 days (long-term immunization regimen, LTIR) of i.v. delivery. A virtually complete recovery was detected after three weeks of 12A12mAb immunization in both novel object recognition test (NORT) and object place recognition task (OPRT). Consistently, three weeks of this immunization regimen relieved in hippocampi from ICV-STZ mice the AD-like up-regulation of amyloid precursor protein (APP), the tau hyperphosphorylation and neuroinflammation, likely due to modulation of the PI3K/AKT/GSK3-β axis and the AMP-activated protein kinase (AMPK) activities. Cerebral oxidative stress, mitochondrial impairment, synaptic and histological alterations occurring in STZ-infused mice were also strongly attenuated by 12A12mAb delivery. These results further strengthen the causal role of N-terminal tau cleavage in AD pathogenesis and indicate that its specific neutralization by non-invasive administration of 12A12mAb can be a therapeutic option for both fAD and sAD patients, as well as for those showing type 2 diabetes as a comorbidity.

T217-Phosphorylation Exacerbates Tau Pathologies and Tau-Induced Cognitive Impairment

Background: Recent studies show that an increased T217-phosphorylation of tau in plasma could diagnose AD at an early stage with high accuracy and high specificity, while the potential toxic role of tau T217-phosphorylation is not known. Objective: To study the potential toxic role of tau T217-phosphorylation. Methods: We performed stereotactic brain injection, behavioral testing, immunohistochemistry and immunofluorescence, western blotting, Golgi staining, in vitro recombinant tau polymerization, and other measurements. Results: We first constructed tau T217-wild-type (T217), T217-phospho-mimic (T217E), and T217-non-phospho-mimic (T217A) plasmids or their virus vectors on the basis of wild-type tau. We found that expressing tau-T217E induced a significantly increased tau phosphorylation at multiple AD-associated sites with inhibited proteolysis and increased cleavage/fibrillization of tau, while expressing tau-T217A abolished the above changes of tau both in vitro and in vivo. By mutating T217E on tau-P301L, a dominant mutation identified in patients with frontotemporal dementia, we did not observe significant exacerbation of tau-P301L phosphorylation and cognitive impairment although the increased tau cleavage and propagation were shown. Conclusion: T217-phosphorylation exacerbates wild-type tau hyperphosphorylation with aggravated tau cleavage/fibrillization and cognitive impairments, while overexpressing T217E on the basis P301L does not exacerbate tau phosphorylation or the P301L-induced cognitive deficits, although it aggravates tau cleavage and propagation.

iPSC-induced neurons with the V337M MAPT mutation are selectively vulnerable to caspase-mediated cleavage of tau and apoptotic cell death

Background: Tau post-translational modifications (PTMs) are associated with progressive tau accumulation and neuronal loss in tauopathies, including forms of frontotemporal lobar degeneration (FTLD) and Alzheimer’s disease (AD). Tau proteolysis by caspases, including caspase-6, represents an understudied PTM that may increase neurotoxicity and tau self-aggregation. Methods: To elucidate the presence and temporal course of caspase activation, tau cleavage, and neuronal death, we generated two novel epitope (neoepitope) monoclonal antibodies (mAbs) against caspase-6 tau proteolytic sites. We evaluated tau cleavage and response to apoptotic stress in cortical neurons derived from induced pluripotent stem cells (iPSCs) with frontotemporal dementia (FTD)-causing V337M MAPT mutation. We tested the neuroprotective effect of caspase inhibitors in the induced neurons. We also demonstrated the presence of the tau neoepitopes in postmortem brains from an individual with FTD (V337M MAPT) and an individual with AD, compared to a healthy control.Results: FTLD V337M MAPT and AD postmortem brains showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau and phosphorylated (p)-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by inhibition of effector caspases.Conclusions: Culturing iPSC-derived neurons for three months exposes age-related tau pathologies, including caspase-mediated cleavage, that are also observed in human postmortem brains with abnormal tau deposition. Neoepitope antibodies to caspase-cleaved tau may serve as biomarkers of tau pathology. Furthermore, caspases could be viable therapeutic targets for tau pathogenesis in FTLD and other tauopathies.

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.

iPSC-Induced Neurons with the V337M MAPT Mutation are Selectively Vulnerable to Caspase-Mediated Cleavage of Tau and Apoptotic Cell Death

Background: Tau post-translational modifications (PTMs) are associated with progressive tau accumulation and neuronal loss in tauopathies, including forms of frontotemporal lobar degeneration (FTLD) and Alzheimer’s disease (AD). Tau proteolysis by caspases, including caspase-6, represents an understudied PTM that may increase neurotoxicity and tau self-aggregation. Methods: To elucidate the presence and temporal course of caspase activation, tau cleavage, and neuronal death, we generated two novel epitope (neoepitope) monoclonal antibodies (mAbs) against caspase-6 tau proteolytic sites. We evaluated tau cleavage and response to apoptotic stress in cortical neurons derived from induced pluripotent stem cells (iPSCs) with frontotemporal dementia (FTD)-causing V337M MAPT mutation. We tested the neuroprotective effect of caspase inhibitors in the induced neurons. We also demonstrated the presence of the tau neoepitopes in postmortem brains from an individual with FTD (V337M MAPT) and an individual with AD, compared to a healthy control. Results: FTLD V337M MAPT and AD postmortem brains showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau and phosphorylated (p)-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by inhibition of effector caspases.Conclusions: Culturing iPSC-derived neurons for three months exposes age-related tau pathologies, including caspase-mediated cleavage, that are also observed in human postmortem brains with abnormal tau deposition. Neoepitope antibodies to caspase-cleaved tau may serve as biomarkers of tau pathology. Furthermore, caspases could be viable therapeutic targets for tau pathogenesis in FTLD and other tauopathies.

Caspase inhibition mitigates tau cleavage and neurotoxicity in iPSC-induced neurons with the V337M MAPT mutation

Tau post-translational modifications (PTMs) are associated with progressive tau accumulation and neuronal loss in tauopathies, including forms of frontotemporal lobar degeneration (FTLD) and Alzheimer disease (AD). Proteolytic cleavage of tau by active caspases, including caspase-6, represents an underexplored tau PTM implicated in tau pathology. Caspase-cleaved tau is toxic and prone to self-aggregation in experimental models. To elucidate the presence and temporal course of caspase activation, tau cleavage, and neuronal death, we generated two neoepitope monoclonal antibodies (mAbs) against caspase-6 tau proteolytic sites and cortical neurons from induced pluripotent stem cells (iPSCs) with the frontotemporal dementia (FTD)-causing V337M MAPT mutation. FTLD V337M MAPT and AD postmortem brains showed positivity for both cleaved tau mAbs as well as active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons showed a time-dependent increase in pathogenic tau in the form of tau oligomers, caspase-cleaved tau, and p-tau. Accumulation of toxic tau species in 3-month V337M MAPT neurons also increased vulnerability to stress, which was pharmacologically rescued by caspase inhibition. We propose a model in which time-dependent accumulation of caspase-cleaved tau in V337M MAPT neurons promotes neurotoxicity that is reversed by caspase-6 inhibition. Caspase-cleaved tau may be a biomarker of tauopathy, and caspases could be viable targets for therapeutic intervention against tau pathogenesis in FTLD and other tauopathies.