Aims

The mechanisms leading to Tau aggregation are still ill-defined, different molecular features have been identified as involved in the aggregation process. The peptide PHF6 (Paired Helical Filament-6, residues 306-VQIVYK-311) is described as a nucleus of Tau aggregation. Different strategies have been proposed as therapeutic approaches in tauopathies, including immunotherapy. However, it remains important to better define the best region(s) of Tau to target , as well as the Tau species (non/phosphorylated, non/soluble or aggregated) and their localization (intra/extra-cellular). In view of this challenge, we have used VHHs (Variable domain of the Heavy- chain of the Heavy-chain-only-antibodies) for their interesting properties and relative ease of generation.

Methods

In partnership with Hybrigenics Company, a synthetic phage display library of VHHs was screened against full-length recTau protein. The epitopes recognized by the selected VHHs, were defined using Nuclear Magnetic Resonance spectroscopy. A VHH targeting an epitope in the microtubule binding domain of tau, corresponding to the PHF6, was selected. Further optimizations of this VHH, using yeast two-hybrid were performed, increasing its intracellular binding capacity and its binding affinity, resulting in a family of VHHs targeting PHF6. These VHHs were next screened for their inhibitory effect towards tau aggregation in vitro, in a cellular model (FRET analysis) as well as in tau transgenic mice.

Results

VHH seem to have the capacity of blocking tau seeding in all cases.

Conclusions

The promising preliminary results of the in vivo open the way for new studies using these VHHs as molecular tools to decipher the best target in Tau immunothérapies.

Aims

Aggregation of pathological tau in the brain coincides with synapse loss and cognitive decline in Alzheimer’s disease. Studies on tauopathy mouse models have shown that pathogenic tau triggers synaptic dysfunction underlying memory deficits, yet it is unclear how the mechanisms that regulate synaptic strength are affected by tau. We previously showed that acetylated tau in Alzheimer’s disease drives the loss of KIBRA, a memory-related protein, from postsynaptic sites which causes impaired long-term potentiation (LTP). Our objectives are to establish whether and how enhancing KIBRA levels can rescue Alzheimer’s disease-related cognitive deficits and pathophysiology in mice with pathogenic tau.

Methods

We generated truncated KIBRA constructs to investigate which KIBRA functional domain is critical for restoring LTP in neurons with pathogenic tau. We next made lentivirus to express the KIBRA C-terminal domain in the hippocampus of transgenic mice with pathogenic human tau to examine its effect on tauopathy-related synaptic plasticity and behavioral impairments.

Results

We found that the C-terminal domain of KIBRA was sufficient to restore LTP and hippocampal-dependent memory in mice with pathogenic tau. The KIBRA C-terminal domain interacts with protein kinase Mζ (PKMζ), a postsynaptic protein that regulates AMPA-type glutamate receptors during synaptic plasticity. PKMζ is required for the KIBRA C-terminal domain rescue of LTP in neurons with pathogenic tau.

Conclusions

Our findings suggest that enhancing levels of KIBRA at synapses can restore synaptic plasticity and memory impairments associated with tauopathy. The mechanism for this effect involves the interaction between KIBRA and PKMζ which modulates the activity-dependent trafficking of AMPA-type glutamate receptors.

Aims

Tauopathies are a group of more than twenty known neurodegenerative disorders that affect millions of people worldwide. There is no cure for tauopathies, and current therapeutic strategies provide limited, late-stage symptomatic treatment. This is partly due to lack of understanding of the molecular mechanisms linking tau and cellular dysfunction, especially during the early stages of disease progression.

Methods

We treated tau transgenic mice with a multi-target kinase inhibitor to identify novel targets that contribute to cognitive impairment and putative therapeutic targets.

Results

Treatment significantly ameliorated neuronal function as determined by behavioral testing and a sensitive imaging technique called manganese-enhanced magnetic resonance imaging (MEMRI) with quantitative R1 mapping. Surprisingly, these benefits occurred despite unchanged hyperphosphorylated tau levels. To elucidate the mechanism behind these improved cognitive outcomes, we performed quantitative proteomics to compare this model with known changes in AD, to identify protein changes similar to known AD alterations calcium, mitochondrial, and metabolic/bioenergetic pathways as highly vulnerable. This is the first characterization of pathway networks altered in this tauopathic mouse model as well as the networks responsive to multi-target kinase inhibition. These analyses reveal pathways associated with cognitive function across tauopathies. We identified disease proteins that are consistent between the tauopathic mouse and human proteome.

Conclusions

Our results further demonstrate the novel therapeutic potential of diminishing nitroxidative stress and highlight its involvement in driving the early stages of neuronal and cognitive dysfunction found in neurodegeneration. Additionally, we identified four proteins displaying a pathological overlap between mouse models of AD and persons with AD.

Aims

Alzheimer’s disease (AD) is pathologically characterized by the accumulation of protein aggregates in the brain, i. e. so called amyloid plaques composed of the β-amyloid peptide (Aß), and neurofibrillary tangles composed of the microtubule-associated protein Tau. Inhibitors of pathological Tau aggregation are attractive for the development of new therapeutic compounds. Two hexapeptide motifs within Tau, designated PHF6* (275VQIINK280) and PHF6 (306VQIVYK311), are known to be essential for tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of tau aggregation. This study focuses on the phage-display selection of D-enantiomeric peptides that bind to the aggregation prone sequence motifs within tau to inhibit pathological tau aggregation. We would like to find out which of the two hexapeptide motifs of Tau is the more promising target for therapy development.

Methods

Employing mirror-image phage display with a large peptide library, we have identified PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. D-enantiomeric peptides are extremely protease stable and considerably less immunogenic than L-peptides. The most interesting peptide and its influence on Tau aggregation was investigated using various biochemical and biophysical methods, and compared to the performance of PHF6-binding peptides selected earlier by our and other groups.

Results

The most promising peptide and its retro-inverso form inhibited PHF6*, Tau^RDΔK280 and full-length Tau fibrilization in vitro. Furthermore, the peptides were able to penetrate cells.

Conclusions

From our preliminary data, it seems likely that PHF6 and PHF6* aggregation inhibitors are comparably effective in inhibiting the aggregation of full-length tau in vitro.

Aims

Alzheimer’s Disease (AD) is characterized by gradual cognitive decline driven by the targeting of synapses by small oligomers of both Aβ (AβO) and tau (tauO), which results in synaptic dysfunction that ultimately underscores disease progression. Indeed, there is ample consensus that targeting oligomer binding to synapses would be an effective therapeutic concept for AD. However, recent failures of clinical trials of Aβ-directed therapeutics suggest reduced effectiveness of targeting Aβ in clinically-manifest AD, redirecting attention onto tau oligomers that are known to increase later in the disease timeline. In support of this vision, here we show that tauO can compete AβO off synapses, thus likely becoming the prevailing toxic species.

Methods

Binding of labeled, pre-formed AβO and tauO onto synaptosomes isolated from human and rodent hippocampus and cortex was evaluated using flow-cytometry and western blot techniques.

Results

We found that tau competes Aβ oligomers off synaptosomes in a dose-dependent fashion, thus becoming the prevailing species associated with the synapses. On the other hand, not only AβO are ineffective in competing tauO off the synapse, but at higher concentration AβO appear to recruit tauO to the synapses. Furthermore, pre-digestion of synaptosome with proteinase K abolishes the ability of tau to compete off Aβ oligomers, suggesting that this phenomenon occurs on a protein substrate

Conclusions

Our results support the concept of tau oligomers becoming the main synaptototoxic species in later AD stages when tauO levels increase dramatically, thus making them an effective therapeutic target.

Supported by NIH/NIA R01AG069433, R01AG060718, R56063405 to GT

Abstract Body

Aims

As the world’s aging population is steadily increasing, novel therapeutics targeting age-related diseases such as Alzheimer’s disease (AD) are of essential and immediate concern. We have developed two therapeutic plasma fractions derived from healthy human donors, GRF6019 and GRF6021, that show beneficial effects on symptoms of aging. GRF6019 has advanced to clinical testing in AD, and GRF6021 is a related plasma fraction that improves cognition, increases neurogenesis and neuronal activity, and dampens neuroinflammation in aged mice. Here, we determined whether GRF6021 could impact AD-specific neuropathology and functional deficits in P301S mutant human tau transgenic mice.

Methods

Six-month-old mice overexpressing human P301S tau were treated with GRF6021. Connectivity and functional brain activity were assessed with functional ultrasound, and cognitive function with behavioral testing. Histology and biochemistry were performed to determine the progression of neuroinflammation and tau pathology.

Results

Transgenic mice exhibited a severe deficit in neuronal activity that was fully rescued with treatment of GRF6021. GRF6021 treatment improved contextual memory in transgenic mice in a fear conditioning paradigm, and histological analysis showed that GRF6021 may induce these benefits by reducing neuroinflammation and tau pathology in the brain.

Conclusions

GRF6021 provides an innovative strategy to modulate multiple parallel mechanisms relevant for aging and AD biology. Treatment of P301S mice with a human plasma fraction ameliorates multiple AD-related symptoms, including deficits in cognition and neuronal activity, microgliosis, and tau pathology. These results demonstrate the potential of plasma fractions to transform approaches and support the advancement of GRF6021 to clinical testing for AD.