Davunetide (also known as AL-108, C201, NAP and NAPVSIPQ (single amino acid letter code)) is the smallest neuroprotective peptide derived from activity-dependent neuroprotective protein (ADNP)1. Davunetide and pipeline products protect nerve cells by associating with microtubule end-binding proteins (EB1/EB3) via the SxIP motif in NAPVSIPQ, thus enhancing microtubule dynamics and Tau-microtubule interaction, in turn, protecting the synapse2–5. We have recently extended davunetide target specificity, showing a Src homology 3 (SH3) domain-ligand association site in NAP (NAPVISP) as being responsible for controlling signaling pathways regulating the cytoskeleton, including actin-Tau interactions6.
At the same time, ADNP shows extensive sexual dichotomy, coupled with davunetide-like correction5. For example, we have shown slower axonal transport in the mouse olfactory nerve as a result of ADNP deficiency, a phenomenon accentuated in males and corrected by application of the NAP EB1/EB3-interacting moiety SKIP2. We have further shown mouse sex-dependent changes in tubulin isotype expression associated with modified ADNP levels and brain maturation2. Additionally, we found higher ADNP mRNA transcript levels in post-mortem male hippocampi, as compared to those in females7. Corroborating studies evaluating traumatic axonal injury in human and rodent neurons, relying on ultra-structural analysis, revealed that female axons were consistently smaller, with fewer microtubules, relative to male axons. Computational modeling indicated that these differences place microtubules in female axons at greater risk than male axons of failure during trauma, leading to neurodegeneration8. Still, clinical trials with davunetide failed to separate women and men. Regardless, davunetide treatment resulted in cognitive score enhancement in amnestic mild cognitive (aMCI)9 and protection of daily living activities in schizophrenia patients9,10. The choice of aMCI as a therapeutic target for davunetide is obvious, as the drug protects against tauopathy3,5 and cell death1, with aMCI being the precursor of Alzheimer’s disease (AD), the major tauopathy. The choice of schizophrenia is equally relevant, as by enhancing microtubule dynamics, davunetide augments the autophagy process, which is deficient in schizophrenia. Importantly, ADNP regulates the autophagy process, interacting with the autophagy-controlling proteins, e.g. microtubule-associated protein 1 light chain 2 (LC2), and regulating beclin 1 expression, with NAP (davunetide) enhancing/replacing ADNP-related deficits and providing neuroprotection11.
Given that tauopathy is a major outcome of ADNP deficiency/pathological mutations in mice5,12 and humans13, and that NAP (davunetide) offered protection against tauopathy in multiple pre-clinical models5,12,14, as well as providing indications of increasing cognitive scores in an aMCI trial9, a clinical study in a pure tauopathy was carried out, with progressive supranuclear palsy (PSP) deemed to be an ideal target for davunetide-mediated protection. However, this study addressed the entire tested population, mixing males and females, as a whole. While showing the safety of the drug, the trial did not meet its endpoints15. In trying to understand these results, one explanation raised the possibility of dosage beyond the bell-shaped dose-response curve, identified in pre-clinical studies1, and further suggested in clinical trials. Thus, in schizophrenia, a 5 mg daily dose was significantly active and a higher 15 mg twice daily dose led to results insignificantly different from when a 5 mg daily dose, as well as the placebo, suggestive of plateauing toward a bell-shaped response, even though the study was under-powered10. In aMCI, there was dose dependence (with the above doses), suggesting disease-specificity. However, the dosage used with PSP (30 mg, twice daily) was two-fold higher than the highest aMCI and schizophrenia dosages10,15 possibly reaching a bell-shaped response.
A search for an alternate explanation for the trial results looked more closely at Tau. Indeed, understanding the regulation of Tau (MAPT) mRNA splicing is important for defining the etiology of PSP, as well as other tauopathies. Tauopathies can be categorized by the presence of Tau aggregates containing 3 (3R) and/or 4 (4R) microtubule-binding domain repeats (determined by inclusion/exclusion of MAPT exon 10) and by the inclusion/exclusion of exons 2 and 3, that translate into two N-terminal Tau domains, with accumulation of 1N4R isoforms in PSP and 0N isoforms in the AD temporal cortex16. Importantly, davunetide/ADNP enhances Tau-microtubule association4. In turn, ADNP directly interacts with the splicing machinery, possibly to suppress MAPT exon 10 inclusion17. Furthermore, while the davunetide trial addressing the 4R tauopathy PSP, a potentially ideal population target for davunetide-mediated protection, was deemed negative15, part of the negative result15 was attributed to preferential NAP-mediated enhancement of the dynamic 3R Tau (i.e., Tau containing 3 microtubule interaction sites), versus the 4R-based Tau-microtubule interaction4.
Lastly, in trying to understand what drives PSP tauopathy, an elegant study identified genetic variations and increases in filamin A in PSP brains. Accordingly, increased filamin A levels enhanced the phosphorylation and insolubility of Tau through interacting actin filaments. In addition, reduced filamin A levels corrected aberrant Tau levels in cells cultured from PSP patients, while transgenic mice carrying human filamin A recapitulated Tau pathology in neurons18. In this respect, we have shown that ADNP contains an actin-binding domain and that NAP corrects actin-associated protein interactions6. At the same time, a filamin A fragment localizes to the nucleus to regulate androgen receptor and co-activator functions, thus implicating sexual dichotomy19. Importantly, gender differences in PSP were not appreciated when the results of the earlier davunetide clinical trial were evaluated15. In contrast, a recent study addressing gender differences in the phenotypic expression and progression of PSP provided a retrospective review of medical records of patients diagnosed with PSP over a 21 year period. This latter study evaluated the interval between disease onset and the presentation of the five clinical disability milestones of PSP, namely, wheelchair dependency, unintelligible speech, severe dysphagia, severe cognitive impairment, and urinary catheterization, to determine disease progression in 334 patients (209 males and 125 females). The study showed that males had an older age at disease onset, with a longer duration of illness at the time of presentation. Tremors were more common, the PSP-Parkinsonism phenotype was more frequent, and the time until wheelchair dependency was earlier in males. In contrast, falls within a year of disease onset, apathy, and executive dysfunction were more frequent and the time until unintelligible speech, severe dysphagia, and cognitive impairment were earlier in females20. Thus, gender differences exist in PSP in terms of clinical characteristics and disease progression, in agreement with the proposed molecular mechanism of the disease.
Taken together with ADNP-mediated regulation of steroid biosynthesis genes13, the estrogen-mediated regulation of alternative splicing21, ADNP binding to actin, and NAP (davunetide) enhancement of actin-Tau interactions6, imply sex differences in PSP patient responses to davunetide, re-evaluated here for the first time. The surprising results showed that davunetide afforded significant neuroprotection to women suffering from PSP.