RESUMEN
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.
Asunto(s)
Enfermedades del Sistema Nervioso Central/etiología , Enfermedades del Sistema Nervioso Central/prevención & control , Diabetes Mellitus Experimental/complicaciones , Fármacos Neuroprotectores/uso terapéutico , Oligopéptidos/uso terapéutico , Animales , Caspasa 3/metabolismo , Enfermedades del Sistema Nervioso Central/patología , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/etiología , Modelos Animales de Enfermedad , Lectinas/metabolismo , Imagen por Resonancia Magnética/métodos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos Mentales/tratamiento farmacológico , Trastornos Mentales/etiología , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Conducta Social , Percepción Espacial/efectos de los fármacos , Sinaptofisina/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Neuropatías Diabéticas/tratamiento farmacológico , Proteínas del Tejido Nervioso/farmacología , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Secuencias de Aminoácidos , Animales , Transporte Axonal , Sitios de Unión , Cognición/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Proteínas de Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/uso terapéutico , Nocicepción/efectos de los fármacos , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/uso terapéutico , Unión Proteica , Ratas , Ratas Sprague-Dawley , Aprendizaje Espacial/efectos de los fármacosRESUMEN
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.
Asunto(s)
Ácidos Aminoisobutíricos/química , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Péptidos beta-Amiloides/toxicidad , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Aprendizaje por Laberinto , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/síntesis química , Oligopéptidos/síntesis química , Fragmentos de Péptidos/toxicidad , Ratas , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Tauopathy, a major pathology in Alzheimer's disease, is also found in ~50% of frontotemporal dementias (FTDs). Tau transcript, a product of a single gene, undergoes alternative splicing to yield 6 protein species, each with either 3 or 4 microtubule binding repeat domains (tau 3R or 4R, associated with dynamic and stable microtubules, respectively). While the healthy human brain shows a 1/1 ratio of tau 3R/4R, this ratio may be dramatically changed in the FTD brain. We have previously discovered that activity-dependent neuroprotective protein (ADNP) is essential for brain formation in the mouse, with ADNP+/- mice exhibiting tauopathy, age-driven neurodegeneration and behavioral deficits. Here, in transgenic mice overexpressing a mutated tau 4R species, in the cerebral cortex but not in the cerebellum, we showed significantly increased ADNP expression (~3-fold transcripts) in the cerebral cortex of young transgenic mice (~disease onset), but not in the cerebellum, as compared to control littermates. The transgene-age-related increased ADNP expression paralleled augmented dynamic tau 3R transcript level compared to control littermates. Blocking mutated tau 4R transgene expression resulted in normalization of ADNP and tau 3R expression. ADNP was previously shown to be a member of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Here, Brahma (Brm), a component of the SWI/SNF complex regulating alternative splicing, showed a similar developmental expression pattern to ADNP. Immunoprecipitations further suggested Brm-ADNP interaction coupled to ADNP - polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF)-binding, with PSF being a direct regulator of tau transcript splicing. It should be noted that although we have shown a correlation between levels of ADNP and tau isoform expression three months of age, we are not presenting evidence of a direct link between the two. Future research into ADNP/tau relations is warranted.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas tau/genética , Animales , Biomarcadores/metabolismo , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Femenino , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Factor de Empalme Asociado a PTB , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismoRESUMEN
NAP (NAPVSIPQ, davunetide) is a microtubule stabilizing peptide drug candidate. Here, we set out to identify NAP-like peptides that provide neuroprotection and reduce tau pathology. NAP-like peptides were derived using publically available search engines, which identified sequence homologies in the microtubule subunit tubulin and in the microtubule associated protein, tau. NATLSIHQ (NAT) and STPTAIPQ were derived from tubulin, and TAPVPMPD (TAP) was derived from tau. All peptides provided neuroprotection against the Alzheimer's disease (AD) toxin, the amyloid-ß 1-42 peptide, although NAT and TAP were much more potent than STPTAIPQ. NAT also protected astrocytes, while STPTAIPQ was active only at micromolar concentrations. Because NAT and TAP were much more potent than STPTAIPQ in neuroprotection, those peptides were also tested for inhibition of tau-like aggregation (the second protein hallmark pathology of AD). Both NAT and TAP inhibited tau-like aggregation, with NAT being active over a very broad concentration range. NAT also protected in vivo in a frontotemporal dementia transgenic mouse model (Tau-Tg), when tested at the age of ~10 months. Results showed significantly decreased levels of the NAP parent protein, activity-dependent neuroprotective protein in the cerebral cortex of the Tau-Tg which was increased back to normal levels by NAT treatment. This was coupled to protection of Brain-Body weight ratio in the compromised Tau-Tg. With AD being the major tauopathy and with tau taking part in frontotemporal dementia, novel NAP derivatives that reduce tauopathy and provide neuroprotection are of basic and clinical interest.