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. 2015 Feb;20(1):109-17.
doi: 10.1038/mp.2014.135. Epub 2014 Oct 28.

Evidence that small molecule enhancement of β-hexosaminidase activity corrects the behavioral phenotype in Dutch APP(E693Q) mice through reduction of ganglioside-bound Aβ

E M Knight  1 , H N Williams  2 , A C Stevens  3 , S H Kim  1 , J C Kottwitz  4 , A D Morant  4 , J W Steele  3 , W L Klein  5 , K Yanagisawa  6 , R E Boyd  2 , D J Lockhart  3 , E R Sjoberg  3 , M E Ehrlich  4 , B A Wustman  3 , S Gandy  7
Affiliations

Evidence that small molecule enhancement of β-hexosaminidase activity corrects the behavioral phenotype in Dutch APP(E693Q) mice through reduction of ganglioside-bound Aβ

E M Knight et al. Mol Psychiatry. 2015 Feb.

Abstract

Certain mutant Alzheimer's amyloid-β (Aβ) peptides (that is, Dutch mutant APP(E693Q)) form complexes with gangliosides (GAβ). These mutant Aβ peptides may also undergo accelerated aggregation and accumulation upon exposure to GM2 and GM3. We hypothesized that increasing β-hexosaminidase (β-hex) activity would lead to a reduction in GM2 levels, which in turn, would cause a reduction in Aβ aggregation and accumulation. The small molecule OT1001 is a β-hex-targeted pharmacological chaperone with good bioavailability, blood-brain barrier penetration, high selectivity for β-hex and low cytotoxicity. Dutch APP(E693Q) transgenic mice accumulate oligomeric Aβ as they age, as well as Aβ oligomer-dose-dependent anxiety and impaired novel object recognition (NOR). Treatment of Dutch APP(E693Q) mice with OT1001 caused a dose-dependent increase in brain β-hex levels up to threefold over those observed at baseline. OT1001 treatment was associated with reduced anxiety, improved learning behavior in the NOR task and dramatically reduced GAβ accumulation in the subiculum and perirhinal cortex, both of which are brain regions required for normal NOR. Pharmacological chaperones that increase β-hex activity may be useful in reducing accumulation of certain mutant species of Aβ and in preventing the associated behavioral pathology.

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Conflict of interest statement

Within the past 5 years, SG has held research grants from Baxter Pharmaceuticals and Amicus Therapeutics. SG is also a member of the Data and Safety Monitoring Board for the Pfizer-Janssen Alzheimer's Immunotherapy Alliance. BAW, ACS, DJL, JWS and ERS are employees or shareholders of OrPhi Therapeutics. HNW and REB are employees and shareholders of Amicus Therapeutics.

Figures

Figure 1
Figure 1
Characterization of β-hex-targeted pharmacological chaperone OT1001. OT1001 is a potent and specific inhibitor of β-hex demonstrating target engagement at sub-micromolar concentrations (a). OT1001 treatment of healthy human derived fibroblasts with OT1001 (95–780 nM) for 5 days resulted in a dose-dependent increase in wild-type total β-hex levels up to threefold (b). Five-week-old male C57BL/J6 mice were given a single 100 mg kg−1 dose of the β-hex-targeted pharmacological chaperone OT1001; plasma (c) and brain (d) OT1001 profiles. A 100 mg kg−1 dose of OT1001 crossed the blood–brain barrier and reached levels in the brain that are predicted to be sufficient to bind and increase β-hex levels (>342 nM) and dropped below Ki (253 nM at pH 5) levels for β-hex within 16 h (n=5 mice per time point). Five-week-old male C57BL/J6 mice were orally gavaged either with vehicle, 30, 100 or 300 mg kg−1 of the β-hex-targeted pharmacological chaperone OT1001 daily up to 14 days. Whole brain β-hex A&S (e) and β-hex B (f) activity (n=5 mice per time point). Data expressed as mean±s.e.m. β-hex, β-hexosaminidase.
Figure 2
Figure 2
Dose-dependent increase in brain β-hex levels following treatment of β-hex-targeted pharmacological chaperone OT1001 in Dutch APPE693Q transgenic mice. Three-month-old male Dutch APPE693Q transgenic mice were either untreated, or orally dosed with vehicle or OT1001 for 3 months. Treatment of OT1001 increased β-hex B (a) and A&S (b) levels in a dose-dependent manner (untreated; n=3, vehicle; n=6, 3 and 10 mg kg−1 OT1001; n=7; for 30 and 100 mg kg−1 OT1001, n=6). Data are expressed as mean±s.e.m. One-way analysis of variance with Bonferroni post hoc analyses, *P<0.05, ***P<0.001. β-hex, β-hexosaminidase.
Figure 3
Figure 3
Reduced anxiety behavior and prevention of onset of learning behavior deficits following treatment of the β-hex-targeted pharmacological chaperone OT1001 in Dutch APPE693Q transgenic mice. Three-month-old male Dutch APPE693Q transgenic mice were either untreated (n=15), or orally dosed with vehicle (n=15) or OT1001 (3, 10, 30 or 100 mg kg−1 OT1001; n=13 per treatment group) for 3 months. Six-month-old vehicle-treated Dutch APPE693Q transgenic mice show increased anxiety relative to 3-month-old untreated mice in the elevated plus maze, which is reversed following treatment with OT1001 at all doses tested (a). Three-month-old untreated mice display intact memory, exploring the novel (new) object more than the familiar (old) object, whereas 6-month-old vehicle-treated Dutch transgenic mice show a learning behavior deficit in novel object recognition (NOR); OT1001 prevents onset of learning deficit in the NOR test in a dose-dependent manner (b). Data expressed as mean±s.e.m. *P<0.05, ***P<0.001. β-hex, β-hexosaminidase.
Figure 4
Figure 4
Aβ-like immunoreactivity was reduced following treatment of the β-hex-targeted pharmacological chaperone OT1001 in Dutch APPE693Q transgenic mice. Sagittal brains sections from male Dutch APPE693Q transgenic mice orally dosed either vehicle (a, c, e) or 100 mg kg−1 OT1001 (b, d, f) for 3 months were stained with the antibody 6E10 (vehicle; n=4, 100 mg kg−1 OT1001; n=4). Aβ immunoreactivity was less intense in the subiculum (a, b) but not visual cortex (c, d) nor in CA1 region of the hippocampus (e, f) of OT1001 treated Dutch APPE693Q transgenic mice. Scale bar, (a–f) 200 μM. β-hex, β-hexosaminidase.
Figure 5
Figure 5
GAβ immunoreactivity was reduced following treatment of the β-hex-targeted pharmacological chaperone OT1001 in Dutch APPE693Q transgenic mice. Sagittal brains sections from male Dutch APPE693Q transgenic mice dosed either vehicle (a, c, e, g, i) or OT1001 (b, d, f, h, j) for 3 months were stained with anti-GAβ (4396C) (vehicle; n=4, 100 mg kg−1 OT1001; n=4). GAβ-like immunoreactivity as detected by 4396C antibody was reduced in the subiculum and perirhinal cortex (k, l). GAβ was unchanged in the visual cortex, lateral entorhinal cortex and CA1 region of the hippocampus (m, n, o). Scale bar, (a–j) 200 μM. GAβ staining in vessels was detectable in the cortical regions (p). Data expressed as mean±s.e.m. Student's t-tests, *P<0.05. β-hex, β-hexosaminidase; GAβ, ganglioside-bound Aβ.
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