Treatment with BSc3094 failed to reverse the body and brain weight loss in rTg4510 mice

The first outcome from the treatment, which was monitored weekly, was the BW of the mice. BSc3094 did not reverse the BW loss typically observed in this transgenic mouse line. Furthermore, at the day of sacrifice, the brain weight was measured. Again, BSc3094 failed to reverse the brain weight loss characteristic in rTg4510 mice.

Previous studies showed that rTg4510 mice present a loss of 60% of CA1 neurons at 5.5 months of age, which results in a significant loss in brain weight (Ramsden et al., 2005; SantaCruz et al., 2005). A likely explanation is that pathological alterations that culminate in this extensive neuronal loss start very early in life, so that it reaches a massive reduction in CA1 cells at 5.5 months of age. Therefore, starting the treatment with BSc3094 at 2 months of age may have been too late to reverse or slow down the neuronal loss characteristic of rTg4510 mice, explaining why BSc3094-treated mice also presented a lower brain weight compared to control littermate mice.

5.3.4. Treatment with BSc3094 significantly reduced the levels of phosphorylated and insoluble tau, and partially improved cognition

Despite this, treatment with BSc3094 strongly reduced the levels of phosphorylated tau (detected with both 12E8 and PHF-1 antibodies), as well as the levels of sarkosyl-insoluble tau, which is in agreement with previous in vitro studies from our group, plus an indicator that pathological hallmarks may be reduced upon BSc3094 treatment.

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In terms of behavioral assessment, treatment with BSc3094 resulted in a reversal of anxiety-like behavior and improvement in certain cognitive tests. Initially, we analyzed locomotor activity in the OF test, to ensure that transgenic mice do not have motor impairments that could lead to differences in the behavioral performance. Although no significant motor impairment was found in rTg4510 mice, we observed an increase in anxiety-like behavior in vehicle-treated transgenic mice, shown by a reduction in the time the mice spent exploring the center of the arena. This phenotype was ameliorated in the BSc3094-treated rTg4510 mice, showing that the drug reduced anxiety-like behavior. Furthermore, BSc3094 treatment reversed the memory impairment in rTg4510 mice in the NOR test, based on the assumption that mice have an innate tendency for exploring a novel environment or object. Vehicle-treated rTg4510 mice spent a significantly lower percentage of time in the new object compared to control mice, showing that they cannot distinguish between the familiar and novel object. On the other hand, BSc3094-treated mice spent a significantly higher percentage of time in the new object compared to the vehicle-treated transgenic mice. Treatment with BSc3094 also improved long-term memory in the MWM maze, as vehicle-treated rTg4510 mice spent a significantly lower percentage of time in the target quadrant during the long-term probe trial (72h after the last training session) compared to control mice. This was partly reversed by treatment with BSc3094, although no beneficial effect was observed on the latency to escape from the water during the MWM learning days or during the other probe trials. Besides this, BSc3094 also failed to reverse the impairments in burrowing and nest building in rTg4510 mice. The treatment with the tau aggregation inhibitor also did not reverse memory deficits observed in the y-maze test, as well as the loss in synaptic markers GluR1, synaptophysin and PSD95 in rTg4510 mice.

Several reasons may explain these conflicting results, including the fact that the different behavioral tasks performed to assess cognition in rTg4510 mice involve different types of memory that require distinct neuronal networks and brain regions for their storage and retrieval (Thompson and Kim, 1996; Ben-Yakov et al., 2015).

Furthermore, the extent of tau pathology and the concentration of BSc3094 reaching each part of the mouse brain may be different, which could contribute to the differences. In addition, the implantation of Alzet osmotic pumps and brain infusion kit in young mice (2 months of age) is a delicate surgery, which may also contribute to an increased variability and the conflicting results. As already mentioned, rTg4510 mice

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develop tangles and memory deficits at a very young age (2.5 months). The levels of mutant htau in this mouse model are also extremely high compared to the levels of endogenous mouse tau (approximately 13 times higher) (Ramsden et al., 2005;

SantaCruz et al., 2005). Based on this, the window for a therapeutical intervention in rTg4510 mice is short. We started the treatment as early as possible, as we wanted to avoid the development of pathological changes in the brain that may be irreversible.

However, we could not start before 2 months of age because, even using the smaller model of Alzet osmotic pumps, the animals needed to weight at least 20 grams to be able to cope with the subcutaneous implantation of the Alzet pump. This may have been a late start for the therapeutic intervention, which further contributes to the conflicting results.

In a future approach, a different mouse model that develops tau aggregates and pathology at a later stage in life could be considered, as this would allow us to perform an earlier interventional approach. Furthermore, it would also facilitate the implantation of the Alzet osmotic pump, as the use of adult animals would prevent certain issues we experienced. Specifically, as the animals were relatively small at 2 months of age, we had problems during and after the implantation of the Alzet pump because the animals were too small and still growing. This led to the skin opening around the cannula, as it was stretched extensively, allowing the mice to remove the Alzet pump or to disconnect the catheter. Another possibility would be to make use of the regulatable expression of the transgene in the rTg4510 mice. Administration of Doxycycline, a tetracycline analog, would lead to the inactivation of the transgene expression, allowing the animals to grow without developing any pathological changes.

This would allow us to perform the BSc3094 treatment later in life, very shortly after stopping the administration of Doxycycline, ensuring that the treatment starts early enough before any pathological changes occur. This would prevent the problems related to the surgery, as older mice could be used.

Another important aspect to mention is the fact that, although BSc3094 treatment resulted in a strong reduction of tau phosphorylation and in the levels of sarkosyl-insoluble tau, there was no robust improvement in terms of memory and cognition. This again suggests that tau aggregates may not be the toxic tau species, but intermediate forms of tau could be causing the pathology and neurodegeneration. Indeed, although inhibiting tau aggregation seems a valid therapeutical strategy for halting tau-mediated

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neurodegeneration in AD, based on the premise that this protein has a crucial role in tauopathies and drives pathology, this assumption has been questioned over the last years. Currently, it is still unclear which form of tau is pathological and if tau aggregates are the toxic tau species or not. If tau aggregates are not deleterious, this means that tau aggregation inhibitors will produce little or no effect, or even be harmful as we may be reducing the tangle load but increasing the number of small oligomers, which are now proposed to be toxic (Cheng et al., 2007; Necula et al., 2007; Kaniyappan et al., 2017).

Due to the multifactorial nature of AD pathogenesis, which involves a host of environmental and genetic factors, it may be necessary to personalize the preventive or therapeutic approach and combine different strategies that target distinct hallmarks of the disease to achieve a therapeutic effect.

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Conclusions

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