Main Research Findings

1) In animal models of induced Alzheimer’s disease, davunetide was found to enhance memory and learning.

2) Intranasal administration of treatment with davunetide was shown to decrease hyperactivity in STOP-knockout mice to protect visual memory and elicit neuroprotective effects against the development of schizophrenia.

 

Selected Data

1) Alzheimer’s disease is typically characterized by the aggregation of a high number of senile plaques. This plaque is composed of the amyloid beta protein, which is found in two different forms: A-beta-1-40, and A-beta-1-42. A-beta-1-42 was found to not only have higher levels of neurotoxicity, but it also aggregates at a faster rate than A-beta-1-40. Additionally, A-beta-1-42 has been shown to damage synaptic transmissions and spatial memory, as well as inhibit long term potentiation driven by the hippocampus. However, further research should be conducted to determine the mechanism of action behind effective neuroprotective compounds, such as davunetide, against the aggregation of amyloid-beta proteins [1].

The research team of Zhang et al utilized 3-4 month old, male Sprague Dawley rats. The test subjects were maintained under standard laboratory conditions and allowed ad libitum access to food and water. All animal procedures included in the study were followed in accordance with the Guidelines for Care and Use of Laboratory Animals. Amyloid beta peptide was synthesized and purity was detected using a reversed-phase high performance liquid chromatography. From there, the A-beta-1-42 solution was prepared by dissolving the compound in 1 mg/ml of 100% HFIP, followed by 1 hour of incubation and sonicating the solution in a water bath for 10 minutes. The resulting compound was divided into small volumes and dried with nitrogen gas in order for storage purposes. DMSO was used to rehydrate the solute prior to treatment, followed by further incubation and vortexing in order to allow for peptide aggregation into oligomers [1].

After the rats were able to acclimate to their conditions for one week, the test subjects were randomly assigned to four experimental groups including: vehicle and saline, vehicle and A-beta-1-42, Davunetide and saline, and Davunetide and A-beta-1-42. All animals were anesthetized and fixed on a stereotaxic instrument while 2 uL either a vehicle or A-beta-1-42 was injected into the rats’ bilateral hippocampi via a micro-injection pump. The day after the surgery, the rats underwent 3 weeks of treatment with either 2.5 ug of davunetide in 10uL saline or 10 uL saline. During the treatment period the rats underwent a series of behavioral tests, as well as in vivo hippocampal long-term potentiation recording.

In order to assess impairments in long term spatial memory the mice underwent the Morris water maze test. The apparatus used for the test was characterized as a circular stainless steel basin with varying cues on a black inner surface. The basin was filled with water and a camera was installed to record the animals’ swimming paths. In order to detect the learning ability of the test subjects, acquisition of the hidden platform was practiced for five consecutive days, 4 times per day. In order to ensure that the rats’ visual and motor functioning were intact the subjects were checked with a visual platform test that included placing the escape platform 2 cm above the water’s surface. The rats were placed in the middle of the maze while a hidden escape platform was placed 1-2 cm below the surface in the first quadrant of the maze. Testing took place twice and trials were limited to 120 seconds [1].

After the behavioral testing occurred hippocampal long-term potentiation took place to assess neuronal plasticity. The rats were anesthetized with 1.3 g/kg of urethane while the research team drilled a 2.5 mm hole into the skull in order to insert stimulation and recording electrodes. The bipolar stimulating electrode was driven downward into the Schaffer collateral/commissural pathway while the recording electrode was placed in the stratum radiatum in the hippocampus CA1 region. Basal fEPSPs were recorded for 30 minutes after being evoked by a continuous single electric stimulus. This procedure was followed by stimulation of long term potentiation of fEPSPs induced by high frequency stimulation. A 30% increase or greater in the amplitude of the fEPSPs after HFS was marked as a successful induction of long term potentiation [1]. fEPSPs were then recorded for 60 minutes in order to determine the potential presynaptic mechanisms involved in the facilitation of long term potentiation.

Following the electrophysiological portion of the study the test subjects were euthanized and the research team dissected and homogenized the bilateral hippocampi of each rat. The homogenates were then centrifuged for 10 minutes and the final total protein concentration was determined using a Micro bicinchoninic acid assay kit. 40 ug of the protein samples were separated by electrophoresis and transferred to PVDF membranes; the membranes were incubated with anti-p-AKT antibodies for 12 hours, followed by rinsing 3 times every 5 minutes with TBST. After incubation with a secondary antibody for 2 hours, the membranes were rinsed an additional three times with TBST. The resulting immunocomplexes were observed using an enhanced chemiluminescence kit [1].

2) Researcher Illana Gozes examined the neuroprotective properties of davunetide, specifically as they relate to schizophrenia and related cognitive behavior. Davunetide is an active fragment of ADNP, which has been shown to play a crucial role in brain formation and development and neuronal plasticity. It has been hypothesized ADNP haploinsufficiency inhibits social and cognitive functions, especially those that are typically impaired with schizophrenia. Microtubules are also vital elements of the cytoskeleton as they provide the scaffolding for neuronal structures and transport systems for various chemical messengers [2]. Individuals with schizophrenia have been shown to have cytoskeletal abnormalities and dysfunctions that tend to arise during development. The structural differences are paired with the abnormal expression of microtubule-associated proteins (MAP2 and MAP5), found in specific fields of hippocampal formation, as well as alterations to the phosphorylation and distribution of MAP1B in varying brain regions.

At the initial breakpoint of a chromosomal translocation related to familial schizophrenia was shown to be driven by the gene disrupted-in-schizophrenia-1 (DISC1). The DISC1 protein is found localized at the centrosome where it assists in regulating the assembly of alpha and beta tubulin into microtubules. When the microtubules are dissociated they assemble from the centrosome. That being said, the cytoplasmic dynein regulator protein nuclear distribution element-like (NUDEL) is crucial to centrosome functioning and neuronal migration during brain development. Disruption of centrosomal or NUDEL functions through the loss of the DISC1 protein has been linked to the developmental abnormalities often reported in schizophrenia [2].

Because the DISC1 protein is an important component of the microtubule-associated dynein motor complex and maintaining the centrosome, expression of carboxy-terminal-truncated mutant DISC1 or depletion of the endogenous DISC1 has been found to impair development of the cerebral cortex in vivo, as well as neurite outgrowth in vitro. Loss of this protein may lead to the neurodevelopmental dysfunction associated with schizophrenia. Additionally, previous studies have found that the kinesin heavy chain member 2 gene (KIF2) may be a gene that is potentially susceptible to mutations related to the development of schizophrenia. This also indicates that kinesin may be implicated in anterograde axoplasmic transport.

Gozes also used immunocytochemical and biochemical approaches that allowed for the identification of DISC1 and the microtubule-interacting protein associated with TNF receptor-associated factor 3, MIPT3, and MAP1A. MAP1A was shown to interact with the N-terminus of DISC 1 as well as stabilize the microtubules expressed in mature neurons. On the other hand, MIPT3 was found to interact with the microtubules and the actin filaments, suggesting that the protein assists in the regulation and maintenance of cytoskeleton structures. Variation in these proteins that lead to the dysfunction of DISC1 affects the structure and function of the hippocampus in a manner that increases the risk for schizophrenia [2].

Allelic and haplotypic linkages to the MAP6 gene have been seen in cases of schizophrenia. The MAP6 gene is typically expressed as two isoforms; upregulation of the mRNA of isoform 2 was observed in the prefrontal cortex in patients with schizophrenia. These findings indicate a potential deregulation of MAP6 may contribute to the disease. The MAP6 protein also acts as a stable tubule-only polypeptide (STOP) that stabilizes the microtubules crucial for brain development and cognitive functioning [2]. Mice with the STOP gene knocked out tend to exhibit synaptic, behavioral, and hyper-motility deficits. Antipsychotic drugs and microtubule stabilizers that enhance synaptic function and behavior have been used previously to protect against cytoskeleton dysfunctions related to schizophrenia. Researcher Illana Gozes examined the peptide davunetide for its microtubule stabilizing properties that may have similar effects against schizophrenia as treatments that are currently utilized [2].

 

Discussion

1) The results of the Morris water maze test revealed that the average escape latency of each subject’s ability to find the hidden platform steadily decreased across each experimental group during the 5 day training period. There was shown to be a significant increase in escape latency in the rats administered A-beta-1-42 on training day 3 and training day 5 in comparison to the rats administered a vehicle. These findings indicate that A-beta-1-42 damaged spatial learning ability in rats. However, davunetide treatment was found to dramatically decrease escape latency on days 3-5, in comparison to the rats administered a vehicle compound and A-beta-1-42.

Following the 5 day trial period, the spatial memory was assessed by removing the platform and allowing the rats to swim for 120 seconds. The results reported that there was a significant relationship between injection of A-beta-1-42 and davunetide. There was also a marked decrease in the amount of time spent swimming in the rats administered a vehicle and A-beta-1-42 in comparison to the rats administered a vehicle and saline [1]. In the group treated with davunetide, there was an obvious recovery that was observed by the research group that was not observed in the group of test subjects injected with A-beta-1-42 and treated with a vehicle compound. It is important to mention that when assessing the motor and visual abilities of the test subjects, the results reported no notable differences between the swimming speed and swimming time to the visible platform. These findings allowed the research team to conclude that administration of both A-beta-1-42 and davunetide did not change the motor or visual abilities of the animals [1].

Following the water maze test the long term potentiation of the hippocampal CA1 region was analyzed in order to assess the association between synaptic plasticity and spatial memory. 30 minutes of baseline fEPSPs were recorded followed by the delivery of 200 Hz impulses into the Schaffer collateral pathway while the fEPSPs from the hippocampal CA1 were recorded for 60 minutes. Statistical analyses performed after the experiment suggested that the injection of A-beta-1-42 injections significantly inhibited the induction of long term potentiation. That being said, davunetide was able to effectively reverse the inhibition of long term potentiation caused by A-beta-1-42. These effects were found to be maintained 60 minutes after the application of 200 Hz high frequency stimulation [1].

 

Figure 1: Changes in A) escape latency, B) time in the target quadrant, C) swimming speed, and D) time to the target in the experimental groups vehicle and saline, vehicle and A-beta-1-42, davunetide and saline, and davunetide and A-beta-1-42.

Additionally, the research team recorded paired-pulse facilitation in order to accurately identify the presynaptic mechanism of action behind long term potentiation and the relationship between davunetide and A-beta-1-42. The paired-pulse facilitation was calculated by comparing the ratio of the second fEPSP over the first fEPSP. There was no significant differences revealed through statistical analysis indicating a mechanism involving the presynaptic neurotransmitters and alpha-beta-1-42 and davunetide administration in the hippocampal CA1 region. These results led the research team to investigate the PI3K/Akt pathway and its involvement in the regulation of cell survival and proliferation, synaptic plasticity, and cognitive performance [1].

In order to determine the association between the regulation of Pi3K/Akt signal pathway and the exact neuroprotective role davunetide plays in synaptic plasticity and spatial memory, Western blotting and two-way ANOVA were utilized. Both statistical analysis platforms suggested that there were no significant effects between AKT and the administration of A-beta-1-42 and davunetide observed in the hippocampal regions. These findings indicate that there were no remarkable changes in the level of Akt proteins. However, when evaluating the levels of pAKT/AKT it was shown that pAKT expression was decreased after the test subjects received an injection of A-beta-1-42, while administration of davunetide significantly reversed these effects [1].

2) ADNP has been found to be an essential protein for the formation of brain tissues. That being said, following the experimental period, researcher Illana Gozes found that mice lacking the ADNP protein experience inhibition of neural tube closure, followed by death at embryonic day 8.5-9.5. Various microarray analyses found that in ADNP knockout mice and embryos from the control group all showed significant changes in the expression of over 400 genes products involved in neurogenesis and embryogenesis, as a result of ADNP deficiency. ADNP expression was downregulated in the pluripotent P19 teratocarcinoma cell line by directing a small hairpin RNA at the ADNP transcript [2]. MAP2 labeling found that an 80% reduction in ADNP resulted in a 50% reduction in neurite numbers, as well as a reduction in embryoid body formation. These findings indicated that there is direct association between the position of ADNP and maturation and differentiation of neuronal cells. This suggests that ADNP may also be associated with neuronal plasticity.

Furthermore, it has been found that in cases of oxidative stress ADNP has the potential to provide neuroprotection; mice experiencing haploinsufficiency had lower levels of astrocytes providing neuroprotection, as well as microtubule associated proteins. In a similar manner to ADNP, ADNP2 is a homologue of ADNP that has also been found to elicit protective effects against oxidative damage due to the 46% similarity between the two compounds [2]. Recent studies evaluating hippocampal specimens using quantitative real time polymerase chain reactions found that there was a remarkable correlation between ADNP to ADNP2 transcript levels. That being said, when observing the hippocampus of matched schizophrenia test subjects the correlation between ADNP and ADNP2 transcript levels was shown to have dramatically decreased. These findings indicate that hippocampal imbalances of the transcript and expression levels may change disease progression of schizophrenia [2].

Additionally, ADNP contains a peptide motif, NAPVSIPQ, generically named davunetide. The motif provides neuroprotective and neurotrophic effects with the capabilities to ameliorate deficiencies related to ADNP levels. Davunetide tends to elicit its neuroprotective effects by interaction with the microtubule cytoskeleton in order to preserve functioning. Related studies have found that treatment of neuronal and glial cells with davunetide leads to the rearrangement of microtubules in a manner that protects against microtubule-zinc intoxication while reversing the microtubule depolymerization induced by nocodazole [2].

In a related manner, mice lacking the microtubule, stable tubule-only polypeptide (STOP) genes were found to act as a reliable model of schizophrenia. Intranasal treatment with davunetide was found to significantly decrease hyperactivity in the microtubule STOP mice and also protected visual memory as seen through the result of the object recognition test where the subjects were indifferent to the novel object and more interested in the familiar object. These findings were in comparison to the control group where the animals were more interested in the novel object. Administration of davunetide also allowed the mice to remember better where the hidden platform was located in the water maze behavioral task. Again, this was in comparison to the STOP knockout mice treated with a vehicle only [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Zhang J, Wei SY, Yuan L, Kong LL, Zhang SX, Wang ZJ, Wu MN, Qi JS. Davunetide improves spatial learning and memory in Alzheimer’s disease-associated rats. Physiol Behav. 2017 May 15;174:67-73. doi: 10.1016/j.physbeh.2017.02.038. Epub 2017 Feb 28. PMID: 28257938.

[2] Gozes I. Microtubules, schizophrenia and cognitive behavior: preclinical development of davunetide (NAP) as a peptide-drug candidate. Peptides. 2011 Feb;32(2):428-31. doi: 10.1016/j.peptides.2010.10.030. Epub 2010 Nov 2. PMID: 21050875.

 

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