D-SAL and NAP: Two Peptides Sharing a SIP Domain.

NAPVSIPQ (NAP) and all D-amino acid SALLRSIPA (D-SAL) are neuroprotective peptides derived from activity-dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF), respectively. Both proteins were shown to protect against cognitive impairment, using different animal models and to increase neuronal survival following exposure to neurotoxins. NAP was extensively tested and found to increase microtubule stability, protect axonal transport, and inhibit apoptosis. Here, we aimed to further evaluate and correlate effects at the behavioral level, in a rat model of diabetes. Diabetes is primarily a metabolic disorder which presents secondary neurological manifestations. Diabetes induces peripheral nervous system damage which is translated into impaired sensory perception and is termed diabetic neuropathy. Diabetes-related central nervous system damage causes cognitive decline. The behavioral study aimed to evaluate the effect of NAP and D-SAL on peripheral neuropathy and cognitive decline. Peripheral neuropathy was tested by measuring the response to a thermal stimulus, and cognitive ability was measured by a social memory test and a spatial memory test using long- and short-term dependent tasks and a reference memory task. Results indicated an immediate sensory neuropathy in the diabetic model, which was prevented by both peptides and a later neuropathic phase, prevented only by NAP treatment. Cognitive tests revealed impaired performance in both social and spatial memory tests in the diabetes model. Each of the peptides improved different aspects of cognitive behavior, with NAP being more potent than D-SAL. Mechanistically, both NAP and SAL contain a SIP (SxIP) domain that has been shown to interact with microtubule end-binding proteins (EBs). Specifically, we have previously shown a direct interaction of NAP with EB1 and EB3; we have further shown an interaction of the NAP-derived 4 amino acid SKIP peptide with EB3, stimulating axonal transport. Interestingly, the all D-amino acid peptide, D-SKIP, only partially mimicked SKIP activity. Our current results implicate D-SAL activity with potentially reduced potency compared to NAP, partially mimicking the SKIP/D-SKIP results and placing the SIP (SxIP) motif as a central focus for microtubule-based neuroprotection.

NAP alpha-aminoisobutyric acid (IsoNAP).

We set out to identify NAP (davunetide) analogs, providing neuroprotection and reducing tau pathology, specifically addressing protection against protein misfolding. NAP (NAPVSIPQ, intranasal formulation AL-108) is a drug candidate that (1) had a statistically significant impact on two measures, namely digit span and delayed-match-to-sample, tests of verbal recall and visual working memory, respectively, in patient population of mild cognitive impairment [preceding Alzheimer's disease (AD)] and (2) protected functional activities of daily living in schizophrenia patients. Previous preclinical studies have shown that stabilization of NAP by replacement of all L-amino acids by D-amino acids resulted in an active peptide, D-NAP. Other NAP mimetics are now explored. A new NAP analog was designed that included replacement of the proline residues by alpha-aminoisobutyric acid to enhance ß-sheet breaker characteristics, thereby reducing protein misfolding. Three lines of investigations were chosen: (1) protection against the AD-associated amyloid ß (1-42), Aß1-42, peptide toxicity in cell cultures; (2) inhibition of AD-associated tau aggregation in vitro; and (3) cognitive protection in a mouse model of deficiencies of the NAP parent protein, activity-dependent neuroprotective protein (ADNP), exhibiting tau pathology and neurodegeneration. NAP alpha-aminoisobutyric acid (IsoNAP) protected neurons against AD-associated Aß1-42-toxicity, inhibited the aggregation of the tau-derived peptide VQIVYK (important for the aggregation of tau into paired helical filaments, which form the tangles found in AD and related disorders), and protected cognitive functions in a model of ADNP deficiency. With AD being the major tauopathy, novel NAP derivatives that reduce tauopathy and provide neuroprotection as well as cognitive protection are of scientific and clinical interest.

Tau and caspase 3 as targets for neuroprotection.

The peptide drug candidate NAP (davunetide) has demonstrated protective effects in various in vivo and in vitro models of neurodegeneration. NAP was shown to reduce tau hyperphosphorylation as well as to prevent caspase-3 activation and cytochrome-3 release from mitochondria, both characteristic of apoptotic cell death. Recent studies suggest that caspases may play a role in tau pathology. The purpose of this study was to evaluate the effect of NAP on tau hyperphosphorylation and caspase activity in the same biological system. Our experimental setup used primary neuronal cultures subjected to oxygen-glucose deprivation (OGD), with and without NAP or caspase inhibitor. Cell viability was assessed by measuring mitochondrial activity (MTS assay), and immunoblots were used for analyzing protein level. It was shown that apoptosis was responsible for all cell death occurring following ischemia, and NAP treatment showed a concentration-dependent protection from cell death. Ischemia caused an increase in the levels of active caspase-3 and hyperphosphorylated tau, both of which were prevented by either NAP or caspase-inhibitor treatment. Our data suggest that, in this model system, caspase activation may be an upstream event to tau hyperphosphorylation, although additional studies will be required to fully elucidate the cascade of events.

Davunetide (NAP) as a preventative treatment for central nervous system complications in a diabetes rat model.

AIMS: Central nervous system complications including cognitive impairment are an early manifestation of diabetes mellitus, also evident in animal models. NAP (generic name, davunetide), a neuroprotective peptide was tested here for its ability to prevent diabetes-related brain pathologies in the streptozotocin injected diabetes rat model. METHODS: Diabetes was induced by an intraperitoneal streptozotocin injection (55 mg/kg). Intranasal NAP or vehicle was administered daily starting on the day following streptozotocin injection. Cognitive assessment was performed 12 weeks after diabetes induction, using the Morris water maze paradigm. Brain structural integrity was assessed on the 15th week of diabetes by magnetic resonance T2 scan. Characterization of cellular populations, apoptosis and synaptic density was performed 16 weeks after diabetes induction, using immunohistochemical markers and quantified in the prefrontal cortex, the cerebral cortex and the hippocampus of both hemispheres. RESULTS: Impaired spatial memory of the diabetic rats was observed in the water maze by attenuated learning curve and worsened performance in the probe memory test. NAP treatment significantly improved both measurements. T2 magnetic resonance imaging revealed atrophy in the prefrontal cortex of the diabetes rat group, which was prevented by NAP treatment. Immunohistochemical analysis showed that NAP treatment protected against major loss of the synaptic marker synaptophysin and astrocytic apoptosis, resulting from streptozotocin treatment. CONCLUSIONS: Our results show for the first time protective effects for NAP (davuentide) in a diabetes rat model at the behavioral and structural levels against one of the most severe complications of diabetes.