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. 2011 Mar;46(3):597-606.
doi: 10.1016/j.mcn.2010.12.011. Epub 2010 Dec 27.

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

Sheila M Fleming  1 , Caitlin K Mulligan, Franziska Richter, Farzad Mortazavi, Vincent Lemesre, Carmen Frias, Chunni Zhu, Alistair Stewart, Illana Gozes, Bruce Morimoto, Marie-Françoise Chesselet
Affiliations

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

Sheila M Fleming et al. Mol Cell Neurosci. 2011 Mar.

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.

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Figures

Figure 1
Figure 1. Effect of NAP administration on motor performance and coordination in Thy1-aSyn and wildtype mice
Errors per step on the challenging beam in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, ** represents p<0.01 compared to respective WT and †† represents p<0.01 compared to Thy1-aSyn Vehicle (2 × 2 ANOVA followed by Fisher’s LSD).
Figure 2
Figure 2. No adverse effects of NAP administration on the challenging beam and pole tests in Thy1-aSyn and wildtype mice
Steps (A) and time to traverse (B) on the challenging beam and time to turn (C) and descend (D) the pole in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, ** represents p<0.01 compared to respective WT (2 × 2 ANOVA followed by Fisher’s LSD (Beam) and Mann-Whitney U (Pole)).
Figure 2
Figure 2. No adverse effects of NAP administration on the challenging beam and pole tests in Thy1-aSyn and wildtype mice
Steps (A) and time to traverse (B) on the challenging beam and time to turn (C) and descend (D) the pole in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, ** represents p<0.01 compared to respective WT (2 × 2 ANOVA followed by Fisher’s LSD (Beam) and Mann-Whitney U (Pole)).
Figure 2
Figure 2. No adverse effects of NAP administration on the challenging beam and pole tests in Thy1-aSyn and wildtype mice
Steps (A) and time to traverse (B) on the challenging beam and time to turn (C) and descend (D) the pole in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, ** represents p<0.01 compared to respective WT (2 × 2 ANOVA followed by Fisher’s LSD (Beam) and Mann-Whitney U (Pole)).
Figure 2
Figure 2. No adverse effects of NAP administration on the challenging beam and pole tests in Thy1-aSyn and wildtype mice
Steps (A) and time to traverse (B) on the challenging beam and time to turn (C) and descend (D) the pole in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, ** represents p<0.01 compared to respective WT (2 × 2 ANOVA followed by Fisher’s LSD (Beam) and Mann-Whitney U (Pole)).
Figure 3
Figure 3. No adverse effect of NAP administration on spontaneous activity in Thy1-aSyn and wildtype mice
Forelimb stepping (A), hindlimb stepping (B), rears (C), and grooming (D) in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, *, ** represents p<0.05, 0.001 compared to respective WT (2 × 2 ANOVA followed by Fisher’s LSD).
Figure 3
Figure 3. No adverse effect of NAP administration on spontaneous activity in Thy1-aSyn and wildtype mice
Forelimb stepping (A), hindlimb stepping (B), rears (C), and grooming (D) in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, *, ** represents p<0.05, 0.001 compared to respective WT (2 × 2 ANOVA followed by Fisher’s LSD).
Figure 3
Figure 3. No adverse effect of NAP administration on spontaneous activity in Thy1-aSyn and wildtype mice
Forelimb stepping (A), hindlimb stepping (B), rears (C), and grooming (D) in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, *, ** represents p<0.05, 0.001 compared to respective WT (2 × 2 ANOVA followed by Fisher’s LSD).
Figure 3
Figure 3. No adverse effect of NAP administration on spontaneous activity in Thy1-aSyn and wildtype mice
Forelimb stepping (A), hindlimb stepping (B), rears (C), and grooming (D) in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, *, ** represents p<0.05, 0.001 compared to respective WT (2 × 2 ANOVA followed by Fisher’s LSD).
Figure 4
Figure 4. No adverse effect of NAP administration on olfaction in Thy1-aSyn and wildtype mice
Latency to find the pellet in the buried pellet (A) and surface pellet (B) test in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, *, ** represents p<0.05, 0.01 compared to respective WT (Mann-Whitney U).
Figure 4
Figure 4. No adverse effect of NAP administration on olfaction in Thy1-aSyn and wildtype mice
Latency to find the pellet in the buried pellet (A) and surface pellet (B) test in WT (Veh=15; NAP=16) and Thy1-aSyn (Veh=12; NAP=13) mice treated with intranasal Vehicle or NAP. Scores are ± SEM, *, ** represents p<0.05, 0.01 compared to respective WT (Mann-Whitney U).
Figure 5
Figure 5. Photomicrograph of Proteinase K-resistant α-synuclein inclusions in the substantia nigra and olfactory bulb in Thy1-aSyn mice
Proteinase K resistant α-synuclein staining in the substantia nigra (SN, A, C and E) and olfactory bulb (B, D and F) of Thy1-aSyn mice treated with vehicle (A, B C and D) or NAP (E and F). The contours of the SN (A) and glomerular layer of olfactory bulb (C) are marked by dotted lines. Higher magnification showing proteinase K resistant α-synuclein aggregates staining in the SN (C and E) and olfactory bulb (D and F) of Thy1-aSyn mice treated with vehicle (C and D) or NAP (E and F). Epl, external plexiform of olfactory bulb; Gl. Glomerular layer of olfactory bulb; ON, olfactory nerve layer. Scale bars in A, 100 μm; B, 200 μm; C-F, 30μm.
Figure 6
Figure 6. Effect of NAP on proteinase K-resistant α-synuclein aggregates in the substantia nigra and olfactory bulb
A, B: Average number (A) and percent surface area occupied by (B) proteinase K-resistant α-synuclein aggregates from two adjacent sections within the substantia nigra in Thy1-aSyn mice treated with vehicle (n=6) or NAP (n=7). C, D: Number (C) and percent surface area occupied (D) by proteinase K-resistant α-synuclein aggregates from one section within the glomerular layer of the olfactory bulb in Thy1-aSyn mice treated with vehicle (n=5) or NAP (n=4). Scores are ± SEM, *, ** represents p<0.05, 0.01 (respectively) compared to vehicle treated Thy1-aSyn mice (Student’s t-test).
Figure 6
Figure 6. Effect of NAP on proteinase K-resistant α-synuclein aggregates in the substantia nigra and olfactory bulb
A, B: Average number (A) and percent surface area occupied by (B) proteinase K-resistant α-synuclein aggregates from two adjacent sections within the substantia nigra in Thy1-aSyn mice treated with vehicle (n=6) or NAP (n=7). C, D: Number (C) and percent surface area occupied (D) by proteinase K-resistant α-synuclein aggregates from one section within the glomerular layer of the olfactory bulb in Thy1-aSyn mice treated with vehicle (n=5) or NAP (n=4). Scores are ± SEM, *, ** represents p<0.05, 0.01 (respectively) compared to vehicle treated Thy1-aSyn mice (Student’s t-test).
Figure 6
Figure 6. Effect of NAP on proteinase K-resistant α-synuclein aggregates in the substantia nigra and olfactory bulb
A, B: Average number (A) and percent surface area occupied by (B) proteinase K-resistant α-synuclein aggregates from two adjacent sections within the substantia nigra in Thy1-aSyn mice treated with vehicle (n=6) or NAP (n=7). C, D: Number (C) and percent surface area occupied (D) by proteinase K-resistant α-synuclein aggregates from one section within the glomerular layer of the olfactory bulb in Thy1-aSyn mice treated with vehicle (n=5) or NAP (n=4). Scores are ± SEM, *, ** represents p<0.05, 0.01 (respectively) compared to vehicle treated Thy1-aSyn mice (Student’s t-test).
Figure 6
Figure 6. Effect of NAP on proteinase K-resistant α-synuclein aggregates in the substantia nigra and olfactory bulb
A, B: Average number (A) and percent surface area occupied by (B) proteinase K-resistant α-synuclein aggregates from two adjacent sections within the substantia nigra in Thy1-aSyn mice treated with vehicle (n=6) or NAP (n=7). C, D: Number (C) and percent surface area occupied (D) by proteinase K-resistant α-synuclein aggregates from one section within the glomerular layer of the olfactory bulb in Thy1-aSyn mice treated with vehicle (n=5) or NAP (n=4). Scores are ± SEM, *, ** represents p<0.05, 0.01 (respectively) compared to vehicle treated Thy1-aSyn mice (Student’s t-test).
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