The neuroprotective peptide NAP inhibits the aggregation of the beta-amyloid peptide
Introduction
Alzheimer’s disease (AD) is one of the most common disorders of the elderly. It affects approximately four million people a year in the United States alone. AD is characterized by intellectual decline described as senile dementia and loss of cognitive skill [41], [48]. Two types of deposit, neurofibrillary tangles and senile plaques characterize the neuropathology of AD. The major component of the plaques is a hydrophobic 39–43 amino acid long peptide that is known as the beta-amyloid peptide (Aβ; [29]). At least in the frontal cortex, elevated Aβ levels precede the formation of neurofibrillary tangles [35]. Aβ is a cleavage product originated by the processing of the plasma membrane protein, the amyloid precursor protein (APP) encoded by a single gene located on chromosome 21 [24]. Several mutations in the APP gene are now known to cause greater tendency towards the formation of plaques and the onset of AD. Down syndrome patients, exhibiting an extra copy of chromosome 21, secrete more Aβ from birth and develop AD by the age of 50 [45], [54]. Moreover, transgenic mice expressing high APP levels gradually develop amyloid plaques in the brain (e.g. [8], [9], [36]). The exact role of APP in the nervous system is not clear but Aβ is secreted constitutively by normal cells in culture, and was detected in the plasma and the cerebrospinal fluid of healthy mammals including humans [53].
Aβ tends to create insoluble deposits around the walls of cerebral blood vessels and extra cellular neuritic plaques interfering with central nervous system cells, thus creating inhibition in cell vitality [42], [44]. Aβ is capable of forming fibrils in vitro, without the addition of other protein. This is affected by different incubation conditions such as: pH, peptide concentration, temperature, etc. [37].
Activity-dependent neuroprotective protein (ADNP) is a recently discovered glial derived component that is augmented by the presence of the widespread neuroprotective peptide, vasoactive intestinal peptide (VIP; [4]). Screening of several peptides derived from ADNP revealed an eight amino acid long peptide, NAPVSIPQ (NAP), that provides neuroprotection from Aβ toxicity. The addition of 25 μM Aβ(25–35), to mixed primary cultures of neurons and glia, led to a decrease of 50–70% in the number of living neurons [15]. Adding NAP (10−16 to 10−15 M) to these cultures provided complete neuroprotection [4]. NAP neuroprotective activities in vitro and in vivo were recently reviewed [19]. For example, NAP exhibited in vivo protection against developmental retardation and learning impairments in apolipoprotein E-deficient mice [4]. In this respect, apolipoprotein E has been suggested as a risk factor for AD and specific apolipoprotein E isoforms are associated with the formation, or protection against the formation of Aβ plaques [8], [16]. Cholino-deficient rats given daily nasal administration of NAP were protected against loss of spatial learning and memory [18]. Furthermore, in a mouse model of closed-head injury, a single injection of NAP protected against edema formation, resulting in accelerated neurobehavioral recovery and reduced mortality [5]. A similar NAP-related neurobehavioral protection was observed in a rat model of stroke [26]. In general, increased APP synthesis leading to Aβ accumulation was associated with head injury and ischemic conditions [5], [25]. The aim of the current research was to explore whether NAP’s protection against neuronal cell death associated with Aβ toxicity is mediated, in part, by direct inhibition of the formation of Aβ fibrils aggregates.
Section snippets
Materials and methods
This work has been approved by the ethics committee of Tel-Aviv University.
Characterization of in vitro Aβ(25–35) and Aβ(1–40) formation
When Aβ peptide fragments (amino acids, 25–35) (100 μM) and Aβ peptides (amino acids, 1–40) (250 μM) were incubated at 37 °C for 7 days and subjected to a ThT fluorimetric assay, an emission signal at 482 nm was observed (Fig. 1, Fig. 2, respectively). This signal was not observed in fresh suspensions of Aβ (not shown).
NAP inhibits Aβ(25–35) aggregate formation
In order to test the ability of Aβ(25–35) to aggregate into beta sheet conformation and the ability of NAP to interrupt fibril formation, aliquots of Aβ(25–35) (100 μM) were incubated
Discussion
The ability of NAP to interfere with Aβ amyloid fibril formation was demonstrated utilizing two specific quantitative methods: a fluorimetric ThT assay and the Synthaloid plate method. Qualitatively, the inhibition of Aβ amyloid fibril formation was also observed at the electron microscope level.
Evaluating the results by fluorimetry revealed that co-addition of NAP to either a solution of Aβ, or to pre-aggregated Aβ fibrils reduced the level of aggregation (fluorescence) formed after several
Acknowledgements
We thank Drs. Mati Fridkin and Avron D. Spier and Mrs. Sara Rubinraut for their invaluable input. We thank Dr. Malcolm Scott for his kind gift of the Synthaloid plates and Dr. Virginia Smith-Swintosky for the scrambled NAP sequence. Supported in part by the Institute for the Study of Aging (ISOA), Allon Therapeutics, Inc., the US–Israel Binational Science Foundation, the Israel Science Foundation and the Lily and Avraham Gildor Chair for the Investigation of Growth Factors.
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