Research ArticleHEALTH AND MEDICINE

Focused ultrasound delivery of a selective TrkA agonist rescues cholinergic function in a mouse model of Alzheimer’s disease

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Science Advances  22 Jan 2020:
Vol. 6, no. 4, eaax6646
DOI: 10.1126/sciadv.aax6646
  • Fig. 1 Intraparenchymal injection of D3, a selective TrkA agonist, but not NGF, increased TrkA-dependent signaling in 6-month-old TgCRND8 mice with NGF and TrkA deficits.

    (A) No change in NGF mRNA levels was found in the MS/DBB and NBM from 4 to 8 months of age in TgCRND8 and non-Tg mice. (B) NGF protein, (C) TrkA mRNA, (D) TrkA protein, and (E) pTrkA levels were reduced in 6- and 8-month-old TgCRND8 relative to non-Tg mice. (F) p75NTR mRNA and (G) p75NTR protein levels remained stable from 4 to 8 months of age in both genotypes. The relative levels of TrkA to p75NTR (H) protein and (I) mRNA in TgCRND8 mice were decreased by 6 months of age compared to non-Tg mice. (J) Fluorescence in situ hybridization revealed down-regulation of TrkA (red) but not p75NTR (white) mRNA transcripts in ChAT+ cholinergic somata (green) of TgCRND8 mice. Scale bars, 20 μm. (K) Representative Western blots from the MS/DBB and NBM after injection of PBS, NGF, and D3 in TgCRND8 mice. D3-treated mice demonstrated increased levels of (L) pTrkA, (M) pAkt, (N) pMAPK, and (O) pCREB relative to NGF- and PBS-injected TgCRND8 mice. (P) pJNK levels were decreased following NGF and D3 treatment compared to PBS-treated mice. There were no changes in total levels of (Q) Akt, (R) MAPK, (S) CREB, and (T) JNK normalized to βtubIII. All mRNA levels were quantified by qRT-PCR. NGF protein was analyzed by ELISA. TrkA and p75NTR protein levels were measured by Western blot. All phosphorylation signal intensity values were normalized to their respective total protein levels. Statistics: Repeated-measures two-way analysis of variance (ANOVA) (A to I) and one-way ANOVA (L to T). Significance: *,^,†P < 0.05; **,††P < 0.01; ***,†††P < 0.001; * indicates comparison to age-matched non-Tg mice; ^ and † indicate comparison with PBS-treated mice. Data represent means ± SEM; n = 8 per group (A to I) and n = 4 per group (L to T).

  • Fig. 2 Noninvasive FUS-induced BBB permeability localized to the basal forebrain under MRI guidance.

    (A) Schematic of the experimental setup. Mice were positioned supine on an MRI-compatible sled with the head coupled to a polyimide membrane with ultrasound gel and placed in a tank filled with degassed, deionized water, housing the transducer and hydrophone. (B) Ultrasound foci in the MS/DBB and NBM regions of the basal forebrain were targeted on (C) axial T2w MR images. (D) BBB permeability was visualized by the influx of an MRI contrast agent (Gadovist) on contrast-enhanced T1w images in the MS/DBB (green arrows) and NBM (purple arrows). (E and F) There were no significant differences in (E) contrast enhancement or (F) pressure required to induce BBB modulation in 6-month-old TgCRND8 mice compared to non-Tg littermates. (G) Histologically, Evans blue dye (red) extravasation was detected in MRIgFUS-targeted regions of the basal forebrain (MS/DBB, top; NBM, bottom) 90 min after sonication. (H) Higher-magnification confocal micrographs in the MS/DBB (top) and NBM (bottom) revealed the entry of Evans blue in the parenchyma of animals treated with MRIgFUS compared to untreated animals, injected with Evans blue without MRIgFUS. ChAT immunoreactivity (green) was used to identify cholinergic cell bodies. Scale bars, 5 mm (C and D), 1 mm (G), and 100 μm (H). Statistics: Student’s t test (E and F). Data represent means + SEM; n = 12 per genotype.

  • Fig. 3 Enhanced D3 delivery and increased NGF protein levels in the basal forebrain following MRIgFUS-induced BBB opening.

    (A) Representative HPLC elution profile corresponding to D3 detection in the basal forebrain following D3/FUS treatment (blue line) relative to PBS/FUS control (black line). a.u., arbitrary units. (B) D3 levels were negligible in the brain after intravenous D3 injection without MRIgFUS (red bars). With MRIgFUS, D3 significantly entered the targeted MS/DBB and NBM in non-Tg and TgCRND8 mice (blue bars). (C) Plasma and serum D3 levels between unsonicated and sonicated animals for both genotypes were comparable. (D) Increases in D3 concentration, and (E) TrkA phosphorylation, were proportional to the contrast enhancement in MRIgFUS-treated regions for both genotypes. (F) NGF protein levels were increased in PBS/FUS- and D3/FUS-treated relative to PBS- and D3-treated animals, respectively. NGF protein levels were decreased in 6-month-old TgCRND8 mice compared to non-Tg littermates, but FUS-induced NGF protein levels were comparable between genotypes. (G) There was a positive correlation between the relative contrast enhancement and NGF levels measured. Statistics: Two-way ANOVA (B, C, and F). Significance: ^,†P < 0.05, **,^^P < 0.01; † indicates comparison with PBS-treated non-Tg mice (genotype effect); ^ indicates comparison with PBS-treated mice of the same genotype (FUS effect); * indicates comparison with D3-treated mice (i.e., intravenous D3, no MRIgFUS) of the same genotype (D3/FUS effect). (D, E, and G) Linear regression analysis. Dashed lines indicate a 95% confidence interval. Data represent means + SEM; n = 4 per group (B to D) and n = 6 per group (E to G).

  • Fig. 4 MRIgFUS delivery of D3 increased TrkA-dependent signaling cascades in the basal forebrain 90 min after treatment.

    (A) Representative Western blots from the MS/DBB and NBM after PBS, PBS/FUS, D3, or D3/FUS treatment. Blots were probed for pTrkA, pAkt, pMAPK, pCREB, pJNK, the corresponding total proteins, and βtubIII to control for loading. Levels of (B) pTrkA, (C) pAkt, (D) pMAPK, and (E) pCREB were reduced in PBS-treated TgCRND8 mice relative to non-Tg mice and stimulated in D3/FUS-treated mice compared to PBS- and D3-treated (no MRIgFUS) controls in the MS/DBB and NBM for both genotypes. (F) Elevated levels of pJNK were found in control TgCRND8 mice relative to non-Tg mice. pJNK was decreased in TgCRND8 mice after D3/FUS treatment compared to control conditions in Tg mice, but levels remained unaltered in non-Tg mice. Statistics: Two-way ANOVA. Significance: *,†P < 0.05; **,††P < 0.01, ***,^^^P < 0.001; † indicates comparison with PBS-treated non-Tg mice (genotype effect); ^ indicates comparison with PBS-treated mice of the same genotype (FUS effect); * indicates comparison with D3-treated group (i.e., intravenous D3, no MRIgFUS) of the same genotype (D3/FUS effect). Data represent means + SEM; n = 6 per group.

  • Fig. 5 MRIgFUS delivery of D3 enhances cholinergic function 3 days after treatment.

    (A and B) ChAT enzymatic activity was significantly reduced in TgCRND8 relative to non-Tg littermates in the (A) MS/DBB and (B) NBM. ChAT activity was significantly increased in D3/FUS-treated compared to D3-treated (no MRIgFUS) controls in the MS/DBB and NBM for both TgCRND8 and non-Tg mice. (C) Basal and potassium depolarization–induced ACh release from hippocampal slices was significantly reduced in TgCRND8 mice compared to non-Tg controls. Evoked hippocampal ACh release was increased after D3/FUS treatment compared to D3-treated controls in both genotypes. Basal release of ACh was not significantly different in D3/FUS-treated compared to D3-treated animals for both genotypes. (D) TgCRND8 mice had decreased levels of basal and potassium-evoked cortical ACh release relative to non-Tg littermates. Evoked ACh release was also elevated in cortical slices from both TgCRND8 and non-Tg mice following D3/FUS treatment, but basal levels of ACh release remained unaltered in both genotypes. Statistics: Two-way ANOVA. Significance: *,†,#P < 0.05; **,††, ##P < 0.01, ***,###P < 0.001; † indicates comparison with D3-treated non-Tg mice (genotype effect); # indicates comparison with basal levels of ACh release of the same treatment and genotype (depolarization effect); * indicates comparison with D3-treated group (i.e., intravenous D3, no MRIgFUS) of the same genotype (D3 /FUS effect). Data represent means + SEM; n = 5 per group.

  • Fig. 6 MRIgFUS delivery of D3, a selective TrkA agonist, to the basal forebrain rescued neurotrophin signaling and cholinergic function in the TgCRND8 model of AD.

    (Left) By 6 months of age, TgCRND8 mice demonstrated deficits in the NGF/TrkA signaling system, similar to what is observed in human AD. TgCRND8 mice had reduced NGF protein levels, TrkA expression, and activation of downstream signaling cascades, coupled with increased JNK activity in the basal forebrain. (Middle) MRIgFUS was used to noninvasively and locally increase BBB permeability in the basal forebrain, enhancing the bioavailability of intravenous D3 to cholinergic cell bodies in the MS/DBB and NBM. Using this therapeutic approach in TgCNRD8 mice, we stimulated TrkA and downstream signaling effectors—pAkt, pMAPK, and pCREB—while simultaneously decreasing pJNK known to be triggered by p75NTR activation. (Right) MRIgFUS delivery of D3 to the basal forebrain led to enhanced cholinergic neurotransmission in widespread axon terminal regions of the CTX and HF, as evidenced by enhanced ChAT activity, the enzyme that synthesizes ACh, and ACh release.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/4/eaax6646/DC1

    Fig. S1. Age-dependent NGF and TrkA deficits in the HF and CTX of TgCRND8 mice.

    Fig. S2. Intraparenchymal injection of D3-induced TrkA-dependent signaling in the basal forebrain of 6-month-old non-Tg mice.

    Fig. S3. Intraparenchymal injection of NGF-stimulated TrkA signaling pathways in the basal forebrain of 6-month-old non-Tg mice.

    Fig. S4. MRIgFUS-mediated BBB disruption in the basal forebrain after intravenous administration of PBS or D3.

    Fig. S5. Increased extravasation of endogenous immunoglobulins into the basal forebrain following MRIgFUS.

    Fig. S6. MRIgFUS BBB disruption in the basal forebrain did not cause erythrocyte extravasation.

    Fig. S7. MRIgFUS-BBB disruption in the basal forebrain did not induce neuronal apoptosis.

    Fig. S8. Western blot analysis of total Akt, MAPK, CREB, and JNK protein levels after MRIgFUS-mediated delivery of D3.

    Table S1. Primer sequences for qRT-PCR.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Age-dependent NGF and TrkA deficits in the HF and CTX of TgCRND8 mice.
    • Fig. S2. Intraparenchymal injection of D3-induced TrkA-dependent signaling in the basal forebrain of 6-month-old non-Tg mice.
    • Fig. S3. Intraparenchymal injection of NGF-stimulated TrkA signaling pathways in the basal forebrain of 6-month-old non-Tg mice.
    • Fig. S4. MRIgFUS-mediated BBB disruption in the basal forebrain after intravenous administration of PBS or D3.
    • Fig. S5. Increased extravasation of endogenous immunoglobulins into the basal forebrain following MRIgFUS.
    • Fig. S6. MRIgFUS BBB disruption in the basal forebrain did not cause erythrocyte extravasation.
    • Fig. S7. MRIgFUS-BBB disruption in the basal forebrain did not induce neuronal apoptosis.
    • Fig. S8. Western blot analysis of total Akt, MAPK, CREB, and JNK protein levels after MRIgFUS-mediated delivery of D3.
    • Table S1. Primer sequences for qRT-PCR.

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