A pilot trial of the microtubule-interacting peptide (NAP) in mice overexpressing alpha-synuclein shows improvement in motor function and reduction of alpha-synuclein inclusions

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Abstract

Abnormal accumulation of α-synuclein is associated with several neurodegenerative disorders (synucleinopathies), including sporadic Parkinson's disease (PD). Genetic mutations and multiplication of α-synuclein cause familial forms of PD and polymorphisms in the α-synuclein gene are associated with PD risk. Overexpression of α-synuclein can impair essential functions within the cell such as microtubule-dependent transport, suggesting that compounds that act on the microtubule system may have therapeutic benefit for synucleinopathies. In this study, mice overexpressing human wildtype α-synuclein under the Thy1 promoter (Thy1-aSyn) and littermate wildtype control mice were administered daily the microtubule-interacting peptide NAPVSIPQ (NAP; also known as davunetide or AL-108) intranasally for 2 months starting at 1 month of age, in a regimen known to produce effective concentrations of the peptide in mouse brain. Motor performance, coordination, and activity were assessed at the end of treatment. Olfactory function, which is altered in PD, was measured 1 month later. Mice were sacrificed at 4.5 months of age, and their brains examined for proteinase K-resistant α-synuclein inclusions in the substantia nigra and olfactory bulb. NAP-treated Thy1-aSyn mice showed a 38% decrease in the number of errors per step in the challenging beam traversal test and a reduction in proteinase K-resistant α-synuclein inclusions in the substantia nigra compared to vehicle treated transgenics. The data indicate a significant behavioral benefit and a long lasting improvement of α-synuclein pathology following administration of a short term (2 months) NAP administration in a mouse model of synucleinopathy.

Introduction

The presynaptic protein α-synuclein accumulates intracellularly in Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy (MSA), diseases collectively known as synucleinopathies. In PD, α-synuclein-containing proteinacious inclusions (Lewy bodies) are present in central and peripheral neurons (Spillantini et al., 1997, Braak et al., 2003). A direct role for α-synuclein in PD pathophysiology is strongly suggested by genetic evidence linking mutations, multiplications, and polymorphisms in the α-synuclein gene with familial and sporadic forms of PD (Cookson, 2009, Pankratz et al., 2009, Simón-Sánchez et al., 2009).

Multiple defects have been observed in cells overexpressing α-synuclein, including impaired endoplasmic reticulum-Golgi vesicular trafficking (Cooper et al., 2006). Microtubules are essential for vesicular movement and overexpression of α-synuclein in cells can lead to the disruption of microtubule-dependent trafficking (Lee et al., 2006). Conversely, impairments within the microtubule complex increase α-synuclein aggregation and toxicity (Kim et al., 2008). In a transgenic model of MSA, with α-synuclein inclusions in oligodendrocytes, α-synuclein binds to β-III tubulin leading to the accumulation of insoluble complexes and neuronal dysfunction, and this accumulation is suppressed by a microtubule depolymerizing compound (Nakayama et al., 2009). Thus, agents modulating the microtubule system may provide therapeutic benefits in synucleinopathies, including their most common form, PD.

NAPVSIPQ (NAP; also known as davunetide or AL-108) is a neuroprotective peptide derived from the activity-dependent neuroprotective protein (ADNP, Bassan et al., 1999) that interacts with both neuronal and glial tubulin to modulate microtubule assembly (Divinski et al., 2004, Divinski et al., 2006, Gozes and Divinski, 2004, Gozes and Divinski, 2007). Importantly, an interaction with β-III tubulin has been suggested (Divinski et al., 2006) and NAP protects neuronal cells in vitro against dopamine toxicity and severe oxidative stress, pathological mechanisms that likely contribute to PD (Offen et al., 2000).

Bioavailability and pharmacokinetic studies after intranasal administration with (3)H-labeled NAP show that it reaches the rodent brain, remains intact 30 min after administration, and dissipates 60 min after administration (Gozes et al., 2000). Similar results were obtained after intraperitoneal (Spong et al., 2001) or intravenous administration (Leker et al., 2002). A liquid-chromatography, mass spectrometry assay demonstrated that intact NAP reaches the brain after either intravenous or intranasal administration, in rat, dog and human. This was reviewed by Gozes et al. (2005) and recently updated to cite Phase II clinical results showing a positive impact on memory function in patients with amnestic mild cognitive impairment, a precursor to Alzheimer's disease, treated with intranasal NAP (davunetide) formulation (AL-108; Gozes et al., 2009). The bioavailability studies were extended to simultaneous measures in cerebrospinal fluid and plasma in rats as well as whole body autoradiography (Morimoto et al., 2009).

Intranasal NAP is also effective against brain pathology in mice. Indeed, NAP has been shown to reduce accumulation of amyloid peptide and, to a greater extent, tau pathology and improve cognitive function in a triple transgenic mouse model of Alzheimer's disease (Matsuoka et al., 2007, Matsuoka et al., 2008). Similarly, in a mouse model of frontotemporal dementia with tau tangle-like formation, intranasal NAP treatment reduced the tau aggregate load (Shiryaev et al., 2009). NAP has also been shown to be beneficial in animal models of stroke (Leker et al., 2002) and fetal alcohol syndrome (Spong et al., 2001). These extensive studies indicate intranasal efficacy in the range of 0.5–30 μg of NAP per mouse (Gozes et al., 2005, Gozes et al., 2009, Matsuoka et al., 2007, Matsuoka et al., 2008 and unpublished data).

Here, we sought to determine whether NAP could improve behavioral and pathological anomalies in a mouse model of synucleinopathy. Mice overexpressing wildtype, human α-synuclein under the Thy1 promoter (Thy1-aSyn) show increased α-synuclein levels throughout the brain (Rockenstein et al., 2002) and develop proteinase K-resistant α-synuclein aggregates in several brain regions, including the substantia nigra and olfactory bulb (Fernagut et al., 2007, Fleming et al., 2008). Young Thy1-aSyn mice show progressive impairments in sensorimotor function and non-motor symptoms that are associated with the pre-manifest and early stages of PD such as deficits in olfaction, autonomic, digestive, and cognitive function (Fleming et al., 2004, Fleming et al., 2008, Fleming and Chesselet, 2009, Wang et al., 2008). By 14 months, these mice show a 40% decrease in striatal dopamine and l-Dopa responsive behavioral deficits, indicating they reproduce canonical aspects of PD (Hean et al., 2010). While dopaminergic deficits occur at older ages, some of the early behavioral and pathological anomalies have high power to detect drug effects and provide powerful endpoint measures to assess potential treatments for α-synuclein-induced cellular dysfunction in younger mice. Here, NAP was tested for efficacy in Thy1-aSyn mice after intranasal administration at a dose known to produce effective drug levels in mouse brain.

Section snippets

Results

In agreement with previous animal and human studies (Gozes et al., 2005, Gozes et al., 2009), the drug treatment was well tolerated throughout the experiment. As previously reported (Wang et al., 2008, Fleming et al., 2004), Thy1-aSyn mice did weigh less compared to wildtype mice but weights did not significantly differ between NAP and vehicle-treated groups. Body temperatures did not differ between genotypes or drug treatment (Table 1). No adverse reaction or difficulty was noted with the

Discussion

In the present study, we show that intranasal administration of the microtubule-interacting peptide NAP for 2 months improves motor performance and coordination and reduces proteinase K-resistant α-synuclein inclusions in the substantia nigra of mice that overexpress wildtype human α-synuclein. NAP administration was well tolerated and had no detrimental effects on weight, body temperature, or behavior. This is the first study to demonstrate a beneficial effect of NAP in a mouse model of

Conclusion

The present study reveals a beneficial effect on sensorimotor function and reduced α-synuclein pathology following a short-term administration of the microtubule-interacting protein NAP. These findings point to a potentially important therapeutic target in PD and to the overall usefulness of the Thy1-aSyn mouse in preclinical drug studies. Given that the microtubule system is essential to overall cellular function it is not surprising that NAP has broad beneficial effects in a variety of

Mice

Animal care was conducted in accordance with the United States Public Health Service Guide for the Care and Use of Laboratory Animals, and procedures were approved by the Institutional Animal Care and Use Committee at the University of California Los Angeles (UCLA). Transgenic mice overexpressing human wildtype α-synuclein under the Thy-1 promoter (Thy1-aSyn) were developed previously and crossed into a hybrid C57BL/6-DBA/2 background (Rockenstein et al., 2002). Animals were maintained on the

Disclosure

AS (VP Commercial Research), BM (VP Drug Development and IG (Chief Scientific Officer) are employed/consult with Allon Therapeutics Inc. which is clinically developing NAP (generic name, davunetide).

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    The present work was supported in part by the National Institutes of Health (National Institute of Neurological Disorders and Stroke) [grant P50NS38367], the Michael J Fox Foundation, Allon Therapeutics Inc., and the Chen Family Foundation. MFC is the Charles H. Markham Professor of Neurology at UCLA and IG is the incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors and the Director of the Adams Super Center for Brain Research at Tel Aviv University.

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