Fig 1: 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.
Fig 2: 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 3: 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 ßtubIII. 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).
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