2.1 FITC-tagging and presence of inhibitor (I) in rat brain
In a previous report, we evaluated a PEGylated block copolymer with side-chain tripeptide-‘LVF’ for its efficacy to inhibit the early aggregation of misfolded Aβ42 peptide and oligomers as well as its capability to degrade the preformed fibrils of Aβ42 in in vitro and in silico models . In this current study, the block copolymer has been reconstructed with a tag, namely fluorescein isothiocyanate (FITC), to trace its presence in rat brain while studying the inhibitor as a potent drug candidate for AD in vivo. The successful tagging with the FITC was confirmed through 1H NMR, UV-Visible spectroscopy, and fluorescence measurement (Supporting Information). Further, the penetration of the FITC functionalized PEGylated block copolymer (Inhibitor, I) inside the rat brain following an intranasal drug delivery was detected through fluorescence spectra, and upon quantification, it was found that 24 nmole, 35 nmole, 18.26 nmole, and 12.78 nmole of I were present at 1 h, 6 h, 12 h, and 24 h respectively after the administration of final dose (Supporting Information, Figure S5, S6). Moreover, the slow rate of fluorescence decline may relate to the persistent efficacy of I. Further, the choice of intranasal drug delivery was worthy in comparison to the intravenous route, as indicated by the insignificant presence of I in rat brain following an intravenous delivery of I (Figure S6c).
2.2 Selecting the therapeutic dose based on acute toxicity
Two effective therapeutic intranasal doses of I were chosen for rats, based on the acute toxicity study. The intranasal LD50 dose of ‘I’ was determined 73 µg mouse dose, equivalent to 3650 µg/kg body weight for a mouse. Based on the LD50 dose, the therapeutic safe doses for rats were selected to 10 µg/10 µl and 20 µg/10 µl for their each nostril (Supporting Information, Table S3 & Figure S7). For this reason, everyday each rat received either 20 µg or 40 µg I, which were nearly 100 µg/kg and 200 µg/kg body weight, respectively.
2.3 Amelioration of cognitive decline and oxidative stress markers in AD rat model
The effect of I on colchicine-induced impairment of learning and memory were assessed by different behavioral studies like open-field and closed-field activity, Y-Maze test, elevated plus maze test (EPM), Step-down latency passive avoidance test, and Morris water maze test.
Open-field activity scores were noted as counts/5 min on day zero before intracerebroventricular (ICV) injection and day 28 after the treatment. The score was decreased to 31.6 from 64.0, to 11.1 from 24.6, and to 5.5 from 13.6 for AD rats as compared to sham on ambulation, rearing, and grooming, respectively. The treated group which received 0.2% (2 mg/ml) of I showed significant improvement by scoring 51.6 (** p<0.01), 18.5 (** p<0.01), and 10.3 (*** p<0.001) as compared to AD on 28 day after treatment in ambulation, rearing and grooming respectively (Figure 1a). Similarly, the gross behavioral activity of the rats was tested through a closed-field study. In this test, the AD group scored 144.6 with respect to the 241.1 of sham on 28 day of the study whereas, the 0.1% and 0.2% treated groups scored 189.5 (** p<0.01) and 206.6 (*** p<0.001) respectively (Figure 1b). Proving a decline in the learning process, the AD rats scored 14.6% (*** p<0.001) spontaneous alteration in the Y-Maze test compared to the 63.1% spontaneous alteration scoring sham group on the 28th day. Interestingly, the 0.1% and 0.2% treated groups scored 27.6% (** p<0.01) and 34.5% (*** p<0.001) alteration showing an improvement in willingness to explore new environment. On testing the memory acquisition on 20th day of colchicine administration through EPM, the colchicine-induced AD rats lost the learning ability significantly, and it took 75 s initial transfer latency (ITL) to enter the closed arm compared to the sham rats, which has taken 40.3 s. The 0.1% and 0.2% treated groups showed ITL of 60 s and 53.16 s, respectively. When retention of the memory was tested on the 21st and 28th day, AD rat has taken 80.5 s for 1st retention transfer latency (1st RTL), and 78.3 s (2nd RTL) with respect to 18.1 s and 15.1 s RTL of the sham rats, respectively. Compared to this, 0.1% and 0.2% treated rats possessed 31.8 s, 28.5 s, 28.3 s, and 24.5 s of 1st and 2nd RTL, respectively. All the EPM data showed a significant difference level with p<0.001 in paired t-test. The passive avoidance paradigm projected the changes in the learning and memory patterns of the rats throughout the experimental period. A probable reason for this may be the inability of the drug candidate to pass the BBB.
During the acquisition trial on the 19th day after ICV injection of colchicine, the sham group has taken 189.3 s of step-down latency (SDL), where they received an electric shock of 20 V on stepping down the platform. The next day this group took more time (226.6 s) of SDL, and finally, their SDL increased to 260.6 s on the 27th day. This signified the spontaneous response of the control group whereas, the AD rats did not show any memory response and stepped down from the platform with decreasing SDL (Figure 1e). Interestingly, the treated groups showed improvement in memory response by consistently taking a long time to step down from the platform with a significance level p<0.001. Lastly, in the Morris water maze test, the spatial navigation ability of the experimental animals was tested, and it was found that the colchicine-induced AD rats showed higher acquisition latency (AL) and retention latency (RL) time than the control group indicating the impaired learning and memory. Also, the improvement of the treated groups in finding the platform within the cut-off time of 3 min imposed the reversal of memory and learning capability as compared to the AD group (Figure 1f).
There are pieces of evidence that acetylcholine-containing neurons contribute significantly to cognitive decline as observed in AD . Acetylcholinesterase (AchE) plays a pivotal role in the metabolism of acetylcholine which acts as a neurotransmitter. Colchicine being responsible for increasing AchE and thereby degenerating cholinergic neurons in AD mimicking experimental animals provides a platform to evaluate the potential AD drugs (Kumar et al., 2007). In this current report, the level of AchE in colchicine-induced rats was significantly high compared to the control group, which got lowered upon treatment with I (Figure 2a). Even higher the dose of I lower was AchE level (31.8%, p<0.001), which was correlated with overall improvement in cognition.
There are reports showing evidence for the correlation between cognitive declines and decreasing oxidative stress markers . Besides evaluating the cognitive functions of experimental animals herein, we have tested the oxidative stress markers viz. lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase activity. Measuring lipid peroxidation marker is the best tool to measure cellular oxidative damage due to oxidative degradation of membrane lipid. In colchicine-induced AD rats, the lipid peroxidation increased by 120% (p<0.001) in the hippocampus compared to the sham group, which upon treatment with 0.1% and 0.2% I got reduced by 23% and 31.5% (p<0.001) (Figure 2b). Similar results were obtained on analyzing the tissue from the cortex (Figure 2b). The cellular antioxidant, namely glutathione in its reduced form (GSH), scavenges free radicals and protects proteins from oxidative damage . Colchicine prompted oxidative stress in Wistar rats by lowering down 65% and 68% total GSH in cortex and hippocampus, respectively, compared to the control rats. Treatment with I improved both cortex and hippocampus’s condition by increasing GSH level 57%, 88%, 49%, and 69% in 0.1% and 0.2% treated rats, respectively (Figure 2c). In addition, GSH, SOD maintains redox balance by channelizing the superoxide radicals into a continuous oxidation/reduction pool . Herein the results implied that colchicine driven reduction (74% and 77% in cortex and hippocampus respectively) in SOD was increased by 84% and 120% in cortex and 94% and 136% in the hippocampus in 0.1% and 0.2% treated rats, respectively (Figure 2d). It has already been proven that catalase functions as the final checkpoint to cellular oxidative damages by converting H2O2 into H2O and O2 . ICV injection of colchicine led to a depletion of catalase by 51% and 56% in cortex and hippocampal area of the brain, respectively, in Wistar rats compared to the control ones. Upon treatment with 0.1% and 0.2% I, these rats exhibited an increase in catalase enzyme both in cortex and hippocampus by 29%, 41%, 37%, and 52%, respectively (Figure 2e). These results indicated the capability of I to improve overall redox balance and oxidative damage induced in Wistar rats by colchicine which mimics the oxidative damage in AD pathology. Importantly, the I-treated control group did not any significant effect in the behavioral and oxidative marker changes, indicating the neutral property of a drug candidate.
2.4 The reversal in neurotransmitters level
Brief profiling of neurotransmitters, mainly dopamine (DA), norepinephrine (NE), and 5-hydroxytriptamine (5-HT) in treated and untreated AD rats, revealed a decrease in AchE, and a significant increase in DA, NE, and 5-HT level in AD versus treated rats was made feasible with the treatment with I. In previous reports, down-regulated dopaminergic neurotransmitters were linked with the pathophysiology of AD . As similar to the previous reports, the dopamine expression was higher in the cortex than the hippocampus in sham rats (Figure 2f) . On exposure to colchicine, the dopamine level in the cortex got decreased in AD mimicking rats (57%) as compared to the sham, which upon treatment with the 0.1% and 0.2% I has been improved by (53% and 70%, Figure 2f). Besides DA, NE level got subsided notably in the hippocampus of AD rats compared to the control ones (Figure 2g). A dose-dependent treatment with I exhibited a 66% and 102% increase in hippocampal-NE level in treated rats, respectively, with respect to the AD ones (Figure 2g). Also, the reduction in 5-HT was modulated 36% and 61% after a dose-dependent treatment of AD rats with 0.1% and 0.2% I, respectively (Figure 2h). Here also, the I-treated control group did not any significant effect in altering the neurotransmitter levels itself. These quantitative progressions in neurotransmitters in the cortex and hippocampus of treated rats were correlated with the cognition improvements throughout the experiment.
2.5 Quantitative amelioration of Aβ42 load in brain tissues
Through immunohistochemical analyses and Congo red staining of the cortex (frontal) and hippocampal brain tissues, it was found that Aβ42 load in the brain, specifically in cortex and hippocampus, was decreased (Figure S8-S11). The decrease in relative fluorescence intensity in immunohistochemistry was 68%, 81%, 71%, and 87% in frontal and hippocampal brain tissues on treating with 0.1% and 0.2% I compared to the AD brain tissue. This signified the lowering of the Aβ42 load indirectly but precisely. Similarly, the Congo red staining of the 0.1% and 0.2% treated frontal and hippocampal rat brain sections showed a 63%, 74%, 63%, and 67% decrease in relative fluorescence intensity, respectively, compared to the AD brain sections.
Also, the brain tissue histochemistry complemented this finding qualitatively (Figure S12). Disorganization, loss, and shrinkage of pyramidal cells and increased vacuolations of the granular cells were prominent in colchicine-treated rat brains (Figure S12b, S12f). Whereas 0.1% and 0.2% treated rat cortex and hippocampal sections were observed with a restoration, preservation, and reduction in apoptosis of small pyramidal cells as well as reduced vacuolations of granular cells. The clumping of pyramidal cells was proof of damage repair (Figure S12d, S12g).
2.6 In vitro analyses of the inhibitor for mitochondrial response in oxidative stressors and neuroinflammation
Encouraged the results obtained in vivo, the in vitro analyses of cellular mitochondrial responses in oxidative stress conditions were appraised. The cellular oxidative damage and its reversal were also observed in astrocytoma U87 cells, which were in complete agreement with the in vivo data. An 81% reduction in ROS generation by I at 1:3 ratio was significant (*** p<0.001) as compared to the Aβ42-treated cells (Figure 3a). Notably, the inhibitor lowered the ROS generation in a dose-dependent manner as compared to the Aβ42 peptide-treated U87 cells (Figure 3a). Also, when tested with the aid of fluorescence activated cell sorter (FACS), the inhibitor-treated cells showed a reduction in ROS generation compared to the Aβ42-treated cells (Figure 3b). These results were in complete agreement with the changes in pro and anti-inflammatory markers, namely TNF-α, IL-6, TGF-β, and IL-10. The ROS generation-induced increase in pro-inflammatory marker TNF-α was significantly high (P<0.001) as compared to the control, which was reduced to 72% when treated with I at 1:1 ratio (P<0.001) (Figure 3c). Similarly, the increased level of IL-6 in Aβ42-treated cells was dropped by 54% when treated with the inhibitor (Figure 3d). Interestingly, the Aβ42-treated U87 cells were coping with the damage repair by increasing the anti-inflammatory markers, viz. TGF-β and IL-10 as compared to the control (Figure 3e, 3f). The treatment with I even enhanced this damage control by increasing the TGF-β and IL-10 levels by 108% and 458%, respectively compared to the Aβ42-treated U87 cells.
The successful monitoring on the levels of inflammatory markers intrigued the survey on mitochondrial membrane potential (MMP) and oxygen consumption rate (OCR) upon treatment with I. Mitochondrial injury and subsequent reduction in MMP was observed in the case of Aβ42-treated U87 cells via the spectral emission shift of JC-1 dye from red to green (Figure 4). Further, an 18% cell population of the inhibitor-treated cells has shown a reversal of MMP compared to the Aβ42-treated U87 cells (Figure 4c, 4d). Moreover, the treatment of Aβ42-treated U87 cells with the inhibitor clearly reflected the improvement in maximal respiration, proton leak, and subsequent ATP production at a significant level in a dose-dependent manner (Figure 5c-5f).
2.7 In silico modeling of the block copolymer and an illustration to drug-mechanism of action
In connection with our previous study, a complete structural dynamics of the PEGylated block copolymer has been obtained in this current report . Previously we reported the tendency of the block copolymer to bind with the Aβ42 fibril, oligomer, and monomer with a hydrophobic competitive inhibition of the ‘KLVFFA’ core of Aβ42. Here a compact 3D structure of the whole block copolymer (with 113 moieties of PEG and 10 units of side-chain tripeptides) has been deciphered running a reasonably long 1.5 µs Molecular dynamics (MD) simulation (Figure S13). Post simulation, an energetically stable structure was acquired (Figure S13b). In terms of the radius of gyration, the compactness of the simulated block copolymer was high (Figure S13c). Moreover, in comparison to the pre-simulated structure, the volume, geometrical diameter, radius, and geometrical shape coefficient of the post-simulated one was relatively low, indicating a better spatial arrangement of all the moieties in the latter one (Table S4). The surface charge distribution was another parameter to judge the stability of a 3D structure. Here the surface charge distribution of the post-simulated block copolymer also proved its stability in this current form (Figure S13d).
To portray the binding interaction of the PEGylated compound with the fibril structure of Aβ42, the compact PEGylated compound was placed randomly within interacting distances (<=5 Å) at various faces of the Aβ42 fibril structure (PDB ID: 5KK3). During each complex (fibril + molecule) MD simulation run for 100 ns, the structural disintegration of the participating unit chain assemblies (Chain A- Chain I and Chain J- Chain R) was observed, and a change in root mean square deviation (RMSD) and inter-unit distance was calculated (Figure 6, S14). The complete disintegration of 9 monomer units (chain A-I and chain J-R) was occurred only when PEGylated compounds were placed on the A and J chain face of the fibril. Thus, a probable mode of interaction of the PEGylated compound that could disintegrate the 9 monomer units (chain A-I and chain J-R) of fibril structure of Aβ42 peptide (PDB ID: 5KK3) was identified in this exercise. This disintegration of the 9 monomer units (chain A-I and chain J-R) of fibril structure of Aβ42 peptide was in complete agreement with the energy utilization by the system and the occurrence of endothermic reaction taken place in isothermal calorimetric (ITC) experimentation in our previously published report .