Farnesyltransferase Inhibitor LNK-754 Attenuates Axonal Dystrophy And Reduces Amyloid Pathology in Mice Part 3

Results

Reduced amyloid plaque burden and tau hyperphosphorylation in the brains of LNK‑754‑ chronically treated 5XFAD mice

LNK-754 and lonafarnib are small-molecule FTIs that have been previously tested in humans in cancer clinical trials and are known to cross the blood-brain barrier [8, 9, 41, 42]. α-synuclein [8] and tau inclusions [9] are reduced by LNK-754 and lonafarnib, respectively, but they have not yet been assessed against the amyloid phenotype of AD. In this study, we investigated their effects on amyloid pathology in the 5XFAD mouse model. 5XFAD mice develop Aβ deposits beginning at 2 months of age [36, 37], and dystrophic neurites are observed at the earliest stages of amyloid deposition [39, 43]. 

The blood-brain barrier and immunity are two very important parts of the human body, and they are inextricably linked. The blood-brain barrier is a protective barrier between the brain and blood that protects the brain from harmful substances from the outside. Immunity is an important mechanism for the human body to resist diseases. So, what is the specific relationship between the blood-brain barrier and immunity?

First, the blood-brain barrier can prevent some harmful bacteria and viruses from entering the brain, thereby protecting the brain from damage. In addition, the blood-brain barrier can also control the permeability of certain substances, allowing them to enter the brain only to a limited extent. This is critical to protecting the health of the nervous system, as too much of certain internal substances entering the brain can cause nerve cell damage.

At the same time, immunity can also play a role in protecting the brain. The human body's immune system can recognize pathogens and harmful substances, and then produce corresponding antibodies to resist their invasion, thus protecting the body from diseases. However, when certain pathogens or substances cross the blood-brain barrier and enter the brain, the immune system responds quickly and produces antibodies to attack and eliminate them.

Therefore, the blood-brain barrier and immunity play a very important role in maintaining human health. The blood-brain barrier prevents harmful substances from entering the brain, while immunity removes pathogens and other harmful substances that bypass the blood-brain barrier and enter the brain. The two work together to ensure the healthy functioning of the brain.

In summary, the blood-brain barrier and immunity cooperate through their respective mechanisms to play an important role in human health. We should focus on protecting their health to ensure the overall health of the body. It can be seen that we need to improve our memory. Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material with many unique effects, one of which is to improve memory. The efficacy of minced meat comes from the many active ingredients it contains, including acid, polysaccharides, flavonoids, etc. These ingredients can promote brain health in a variety of ways.

Click know supplements to improve memory

Therefore, to coincide with the development of amyloid pathology in 5XFAD mice, we treated mice chronically for 3 months with the vehicle, LNK-754, and lonafarnib, beginning at 2 months of age. LNK-754 and lonafarnib were dissolved in a vehicle of 0.5% sodium carboxymethylcellulose and i.p injected daily with a 1mg/kg dose [8, 44].

We first sought to determine the effects of chronic LNK-754 and lonafarnib treatment on Aβ plaque burden in 5XFAD mice by immunofluorescence microscopy for Aβ42 which detects both diffuse and aggregated amyloid and Tiazine Red (TR), which detects only fibrillar plaque cores (Fig. 1A; Fig. S1). We found a significant decrease in the covered area in cortical brain regions of LNK754-treated mice compared to vehicle-treated controls (Fig.  1B). In the hippocampus, the covered area was significantly decreased in LNK-754-treated mice compared to lonafarnib-treated mice, and there was a trend toward a decrease in LNK-754 treated mice compared to vehicle-treatment (Fig.  1C).

Aβ42 covered area in lonafarnib-treated mice was not significantly different from vehicle-treated mice in either the cortex or hippocampus (Fig.  1A, B). Plaque size was decreased in cortical and hippocampal brain regions by LNK-754 treatment compared to vehicle treatment; this difference was statistically significant in the hippocampus (Fig. 1D, E; Fig. S2A, B). A significant difference in plaque size between lonafarnib and LNK-754 treatment was also observed in the hippocampus (Fig.  1D, E; Fig. S2A, B). 

Plaque size was unchanged in lonafarnib-treated mice compared to vehicle-treated mice in cortical or hippocampal brain regions (Fig. 1D, E; Fig. S2A, B). Aβ plaque seeding was quantified by analyzing plaque number by TR staining. Plaque number significantly decreased in the cortex, but not the hippocampus, of LNK-754 compared to vehicle-treated mice (Fig. S2C, D). Reduced plaque burden was confirmed by measuring Aβ42 by ELISA in PBS and guanidine-soluble hemibrain homogenates (Fig. 1F, G). Similar to our immunofluorescence microscopy results, there was a trend towards less Aβ42 in guanidine and PBS fractions in lonafarnib-treated mice, whereas a significantly lower level of Aβ42 in guanidine fractions was achieved with LNK-754 treatment (Fig. 1. F, G).

Since sex-based effects in the 5XFAD mouse model have been reported for steady-state APP levels and plaque burden [45, 46], we performed immunoblots of male and female mice treated with vehicle, LNK-754, or lonafarnib separately for transgenic APP expression (Fig. S3A). Transgenic APP levels were measured by immunoblot with the APP 6E10 antibody, which recognizes only human APP, and were unchanged in male or female mice treated with LNK-754, lonafarnib, or vehicle (Fig. S3B-D). A second defining feature of AD is the presence of hyperphosphorylated tau inclusions in the brain and along with plaque deposition, endogenous mouse tau is hyperphosphorylated in 5XFAD mice [47, 48]. 

To determine if tau hyperphosphorylation was affected by FTI treatment in an amyloid mouse model, immunoblot analysis was performed for phospho-tau at serine 404 (Fig. 1H; Fig. S3E), an epitope known to be hyperphosphorylated in AD brains and 5XFAD mice [47, 49]. Here, we found reduced phospho-tau ser404 levels in the brains of lonafarnib and LNK-754 treated mice (Fig. 1I), while the total tau level remained unchanged (Fig.  1J; Fig. S3F). Altogether our results reveal that amyloid plaque load and pathogenic tau phosphorylation are reduced in the 5XFAD mouse model by pharmacological treatment with FTIs, although LNK-754 treatment was considerably more potent than lonafarnib.

Chronic treatment with LNK‑754 inhibits protein farnesylation in the brains of 5XFAD mice

To confirm that targeting FTase with LNK-754 and lonafarnib by i.p. injection inhibited protein farnesylation in the brains of 5XFAD mice, we evaluated various FTase substrates and downstream markers of FTase inhibition in hemibrain homogenates of 5XFAD mice chronically treated with vehicle, LNK-754 or lonafarnib by immunoblot analysis. We first analyzed prelamin A, which accumulates when FTase is inhibited [50]. An increase in prelamin A was observed in the brains of mice treated with LNK-754 compared with vehicle, while no change occurred in lonafarnib-treated mice (Fig. S4A, D). We next looked at HDJ-2, a chaperone protein that undergoes an upward mobility shift following FTI treatment and is widely used as a marker of FTase inhibition [50]. Consistent with prelamin A, a slower migrating form of HDJ-2 was present in LNK-754-treated mice but not in lonafarnib-treated mice (Fig. S4B, E). A significant increase in the slower migrating species of HDJ-2 was found in LNK754 mice compared to vehicle or lonafarnib-treated mice (Fig. S4E). 

In addition, farnesylation activates Ras, so we also measured the phosphorylation of its downstream effector molecule ERK1 (P44/P42) (Fig. S4C). Whereas HDJ-2 and prelamin A were unchanged by the lonafarnib treatment, a trend towards reduced pERK1 was observed (Fig. S4F). pERK1 was also decreased in mice treated with LNK-754 (Fig. S4F). Our results suggest that 1mg/ kg LNK-754 inhibited FTase in the brain, however, lonafarnib was less effective. This could be due to the lower potency and IC50 of lonafarnib as shown previously [41, 51], or possibly reduced brain exposure.

Decreased LAMP1 accumulation in dystrophic neurites in LNK‑754 chronically treated 5XFAD mice

In healthy neurons, lysosomal components are delivered to endosomes and autophagic organelles in distal neurites, which become acidifed and mature during retrograde trafficking toward the neuronal soma [52, 53]. Amyloid plaques disrupt this process, resulting in dystrophic neurites that entrap dysfunctional autophagic and endolysosomal organelles. 

Dystrophic neurites block the axonal transport of BACE1, leading to Aβ generation within dystrophic neurites and extracellular plaque deposition [16–18]. FTIs are known to enhance lysosomal function and microtubule stability, so to further investigate how FTIs reduce plaque burden, we focused specifically on dystrophic neurites in 5XFAD mice.

To quantify the accumulation of dystrophic neurites around plaques, we evaluated lysosomal-associated membrane protein 1 (LAMP1), a marker of heterogenous endolysosomal and autophagic organelles. Hippocampal and cortical brain tissues from 5XFAD mice treated with vehicle, LNK-754, and lonafarnib were co-stained for LAMP1 and Aβ42, and confocal immunofluorescence microscopy was performed (Fig.  2A, B).
A threshold was set to eliminate LAMP1 fluorescence signals from vesicles in healthy neurites and somas to specifically analyze dystrophic neurites. LAMP1 covered area was significantly decreased in cortical and hippocampal brain regions of LNK-754 treated mice (Fig. 2C, D), whereas no difference was observed in lonafarnib-treated mice compared to vehicle treatment (Fig.  2C, D). The deposition of amyloid plaques initiates the development of dystrophies in nearby neurites, mainly axons and presynaptic terminals. 

Correspondingly, the effects of LNK-754 and lonafarnib on dystrophic neurites are consistent with their effects on plaque burden (Fig. 1). Additional analysis of the effect of FTI treatment on dystrophic neurites was performed by quantifying the ratio of LAMP1 covered area to Aβ42 area to determine whether FTIs constrain dystrophy size around plaques. A decrease in the ratio of LAMP1: Aβ42 was observed for LNK-754 treated mice compared to vehicle mice, and LNK-754 and lonafarnib treatments were significantly different (Fig. 2E, F), indicating that LNK-754 mice associated with fewer dystrophic neurites.
Elevated levels of LAMP1 were found in hemibrain homogenates of mice treated with either LNK-754 or lonafarnib (Fig. S5A, B), consistent with previous studies showing that lonafarnib and LNK-754 increase the number of autolysosomes and levels of lysosomal membrane proteins and hydrolases [8, 9]. In agreement with immunoblot analysis results for LAMP1 (Fig. S5A, B), the mean fluorescence intensity of total LAMP1 immunostaining was significantly increased in the brains of LNK-754 and lonafarnib-treated mice compared with vehicle-treated mice (Fig. S5C, D). 

In contrast to the total LAMP1 fluorescence level, the mean LAMP1 fluorescence intensity in dystrophic neurites was unchanged between vehicle, LNK-754, and lonafarnib-treated mice (Fig. S5E, F). In the cortex of LNK-754 treated mice, LAMP1 fluorescence intensity in dystrophies and plaque burden were highly correlated, while no correlation between LAMP1 intensity in dystrophies and plaque burden was found in lonafarnib or vehicle-treated mice (Fig.  2G, H), revealing a connection between dystrophic neurite LAMP1 accumulation and plaque burden in LNK-754 mice. A trend towards a correlation between LAMP1 intensity in dystrophies and plaque burden was observed in the hippocampus. Together, our results demonstrate that LNK-754 treatment increases the total level of LAMP1 yet attenuates the pathological accumulation of LAMP1 in dystrophic neurites that are usually present in 5XFAD mice.

Decreased BACE1 accumulation in dystrophic neurites in LNK‑754 chronically treated 5XFAD mice

LAMP1 is found on membranes of endolysosomal organelles that target axonal BACE1 to mature lysosomes for degradation. Therefore, FTIs could inhibit Aβ generation and extracellular plaque deposition by reducing axonal dystrophy formation and the accumulation of BACE1 around plaques. In support of this hypothesis, dystrophic neurite LAMP1 level and plaque burden were highly correlated in cortical brain regions in LNK-754 mice. 

Our analysis of LAMP1 immunostaining revealed that, compared to vehicle or lonafarnib treatment, plaques in LNK-754 treated 5XFAD mice were associated with fewer dystrophic neurites (Fig. 2). To determine whether the level of BACE1 in dystrophic neurites was similarly decreased per plaque, we performed confocal immunofluorescence microscopy of cortical and hippocampal brain tissues of vehicle, LNK-754 and lonafarnib treated mice for BACE1, in addition to LAMP1 and Aβ42 (Fig. 3A, B). 

Here, BACE1 covered area was significantly decreased in cortical brain regions of LNK-754 treated mice, and a trend was observed in lonafarnib-treated mice (Fig.  3C). Te analysis of BACE1 covered area in dystrophic neurites in the hippocampus was impeded by the high expression level of BACE1 in presynaptic terminals of the CA3 hippocampal mossy fiber pathway [17]. We additionally analyzed whether less BACE1 localized to endolysosomes in dystrophic neurites by measuring Pearson’s correlation coefficient for BACE1 and LAMP1 in dystrophies. A significant decrease in BACE1 and LAMP1 co-localization per plaque was found in cortical brain regions in LNK-754-treated mice compared to vehicle treatment (Fig.  3D). 

In the hippocampus, a significant decrease in BACE1 and LAMP1 co-localization per plaque was found for LNK-754 compared to lonafarnib, and a trend towards a decrease was observed for LNK-754 treatment compared to vehicle (Fig. 3E). Tese results are consistent with our findings for plaque burden and LAMP1 covered area, and further support the idea that LNK-754 treatment attenuated the formation of dystrophic neurites, reducing the buildup of impaired endolysosomal organelles that target BACE1 for degradation.

Acute treatment with LNK‑754 reduces late endosome and lysosome accumulation in dystrophic neurites in 5XFAD mice live brain slices

We next tested the acute effects of LNK-754 and lonafarnib on dystrophic neurites by performing confocal imaging of live brain tissues. In this experiment, 5-month-old 5XFAD mice were injected daily with vehicle, LNK-754, or lonafarnib for 3 weeks. We stained tissues with LysoTracker-Green (LT), a probe commonly used for tracking and selectively identifying acidifed late endosomes and lysosomes in living cells [53]. The pattern of LT and thiazine red staining was consistent with our immunostaining results for dystrophic neurite markers in 5XFAD mice (Fig.  2A, B; Fig.  3A, B); LT-positive late endosomes and lysosomes were highly concentrated in dystrophic neurites closely associated with amyloid plaques in living brain tissues (Fig. 4A). 

An acute 3-week treatment of 5-month-old 5XFAD mice with LNK-754 or lonafarnib was insufficient to reduce plaque size (Fig. 4B), although LNK-754 treatment caused a significant decrease in the ratio of LT: TR per plaque and dystrophic neurite size (Fig. 4C, D). Interestingly, in contrast to our findings for LAMP1 or BACE1, acute lonafarnib treatment also decreased dystrophy size and the ratio of LT: TR per plaque (Fig. 4C, D). Dystrophic neurite size and plaque size highly correlated in LNK-754 treated 5XFAD mice (Fig.  4F), and a significant correlation was also observed in lonafarnib-treated mice (Fig. 4G). No correlation existed in vehicle-treated mice (Fig. 4E). Our results confirm that LNK-754 decreased axonal dystrophy and suggest that FTIs can attenuate late endosome and lysosome accumulation around plaques, even after extensive plaque deposition and axon degeneration.

Improvement of learning and memory deficits in hAPP/PS1 mice after short‑term treatment with LNK‑754

LNK-754 prevented the accumulation of LAMP1, BACE1, and LT in dystrophic neurites in 5XFAD mice, suggesting that LNK-754 treatment protects against Aβ-associated synaptic dysfunction. To evaluate whether LNK-754 treatment had a beneficial effect on cognitive function, we assessed the short-term efficacy of oral LNK-754 treatment on spatial learning and memory in the hAPP/ PS1 AD mouse model [40]. Spatial memory of hAPP/ PS1 mice was improved after 12 days of oral treatment with LNK-754, as assessed by the Morris Water Maze (MWM). 

During the hidden platform training sessions, LNK-754 treated hAPP/PS1 mice learned to locate the platform faster than vehicle-treated hAPP/PS1 mice, which resulted in a learning curve that is significantly different from that of vehicle-treated hAPP/PS1 control mice (Fig. 5A). Vehicle-treated hAPP/PS1 mice took significantly more time to locate the platform compared to vehicle-treated wild-type controls on days 4 and 7 of the training trials, whereas no significant differences were found between LNK-754 treated hAPP/PS1 mice and vehicle-treated wild-type mice (Fig.  5A-C). 

During the probe test, LNK-754-treated hAPP/PS1 animals showed increased time spent in the target quadrant (Fig. 5D) and increased frequency of platform crossings (Fig. 5E), comparable to vehicle-treated wild-type animals. Aβ42 levels were measured by ELISA and were unchanged in soluble and insoluble brain homogenates from vehicle-treated or LNK-754-treated hAPP/PS1 mice (Fig.  5F, G), suggesting that improved memory and spatial learning were not a result of lowered amyloid burden in LNK-754 treated hAPP/PS1 mice.

For more information:1950477648nn@gmail.com