Synthesis of polycyclitols
In order to understand and address the issues of weak and non-specific binding of scyllo -inositol with the target proteins, more efforts are required to be channelized in this direction. One possible way-out is to keep the primary backbone of inositol intact and increase the bulk around the molecule by either stitching certain bulky substitutions or more reliably create some related molecules through incisive synthetic interventions like ring annulated conduritol 7 , annulated inositols (8 -10 ) or even fully functionalized inosito-inositols (11-12 ), Fig. 2. Our synthetic endeavour was envisioned from the diepoxide (13 ) which in turn was synthesized from commercially available naphthalene through a well-established reaction sequence (Rashid et al., 2020). Stereo- and regioselective opening of α-epoxide ring in (13 ) under mild acidic conditions led to epoxydiol (14 ). Cis-dihydroxylation of the double bonds with OsO4/NMMO and base mediated carbonate deprotection furnished the annulated inositol (15 ). Under stronger acidic conditions and extended reaction times, both the epoxide rings of diepoxide (13 ) cleaved regioselectively to furnish tricyclic tetrol (16 ). Concurrent dihydroxylation of both the double bonds resulted in the carbonate protected octol (17 ). Deprotection of the carbonate group in (17 ) delivered the target inositol-inositol (18 ) in excellent yield (Fig 3).
SR4 (1-4) derivatives as adjunct therapy to ARM, restored neurobehavioral parameters in ECM
RMCBS is behaviour scale for evaluating the symptoms of CM (Carroll et al., 2010). Animals post infection with PbA exhibited a decreasing trend in the RMCBS score from day 5. Animals symptomatic to CM showed crouching, piloerection, seizures, and retinal haemorrhage on day 7 with RMCBS score of 6.33±0.88. We observed a significant increase in total RMCBS scores in animals administered with polycyclitols (SR4-01, 02, 03, 04) along with ARM (SR4-01; day 5 (16.33±0.66), day 6 (12.66±1.45), day 7 (11.00±1.15), day 8 (14.33±0.33), day 9 (17.33±0.33) and day 10 (17.00±0.57) SR4-02; day 5 (16.66±0.33), day 6 (12.66±0.88), day 7 (16.0±0.57), day 8 (18±0), day 9 (17±0.57) and day 10 (17.66±0.33), SR4-03; day 5 (17.0±0.57), day 6 (14.0±0.57), day 7 (12.0±0.57), day 8 (16.0±0.57), day 9 (16.3±0.88) and day 10 (17.33±0.33) SR4-04; day 5 (16.33±0.33), day 6 (15.0±0.57), day 7 (12.66±0.33), day 8 (16.66±0.33), day 9 (17.66±0.33) and day 10 (16.33±0.33) (p<0.001) compared to CM group (Fig 4A). All SR4 (01-04) adjunctive treated animals showed better locomotion and rearing behavior with an increasing trend from day 8 to 10. After day 30 of adjunctive therapy, SR4-03 exhibited a significant increase in the survival rate of 66 percent (p<0.001) compared to SR4 (01, 02 and 04) treated animals (50 percent) (Fig 4B). Few rescue treated animals died during the survivability phase. Animals were subjected to cognitive tests after day 30 (Fig 4C). As per the guidelines, a 4-5 day interval period was maintained between each cognitive test to minimize error rate.
Polycyclitol adjunctives restored cognitive function in ECM
Accumulation of hyperphosphorylated tau Ser396 in hippocampal CA1 region is associated with inducing neurotoxicity, promoting spatial learning and memory dysfunction correlating to cognitive decline (Hyman et al., 1990; Regalado-Reyes et al., 2019; Scheff et al., 2006; West et al., 2004). We observed that SR4-02 and SR4-04 treated animals performed primary latency significantly within a shorter time interval (SR4-02; 27±3.42 sec on day 1, 19.0±6.3 sec on day 2, 16.23±7.3 sec on day 3, 9.9±6.89 sec on day 4 and 7.32±7.46 sec on day 5 (probe trail), SR4-04; 35.57±8.6 sec on day 1, 31.74±7.8 sec on day 2, 22.45±6.8 sec on day 3, 16.75±7.2 sec on day 4 and 13.56±7.2 sec on day 5 (probe trail)) (p<0.001) compared to SR4-01 and SR4-03 (p<0.001) (45.0±5.56 sec on day 1, 40.0±7.96 sec on day 2, 51.6±6.21 sec on day 3, 43.87±7.9 on day 4 and 46.78±7.43 sec on day 5 (probe trail)) and SR4-03 (68.00±8.56 sec on day 1, 54±7.89 sec on day 2, 22.45±6.8 sec on day 3, 16.75±7.2 sec on day 4 and 13.56±7.2 sec on day 5 (probe trail)) in the Barnes Maze experiment (Fig 5A). The error rate on probe trial day was significantly decreased in SR4-02 (1.8±0.01) (p<0.001) and 04 treated groups (1.9±0.2) compared to SR4-01 (19.3±3.2) and SR4-03 (3.45±2.45). There was no significance in the error rate between SR4-03 and SR4-04 (p=0.593). Frozen behavior was observed especially in SR4-03 treated group (Fig 5 B and D).The heat maps and track plots show that SR4-02 and SR4-04 treated groups spent more time near the escape platform than SR4-01 and SR4-03 (Fig-5 C and E). The CON group was taken as a reference which exhibited primary latency at the earliest time point (18.12±1.2 sec on day 1, 14.12±4.10 sec on day 2, 7.86±3.8 sec on day 3, 4.12±2.89 sec on day 4 and 6.17±0.98 sec on day 5 (probe trail) with a negligible error rate (0.12±0.01). Overall, SR4-02 and SR4-04 experimental groups exhibited a better spatial reference memory with low error rate compared to SR4-01 and SR4-03. In retrieval phase of novel-object recognition test, the track plots revealed that SR4-02 treated group showed a significant increase in the time spent with the novel object (SR4-02: 147±2.5 sec) to the rest of the groups (SR4-04:140±0.5 sec (p=0.044), SR4-03: 127.5±7.5 sec (p<0.001) and SR4-01: 137.5±7.5 sec (p=0.022).We observed that SR4-02 group significantly spent least time with the known object compared to (SR4-01:130.0±10 (p<0.001), SR4-03:112.5±2.5 sec (p=0.001), SR4-04: 109±4.5 (p=0.002) (Fig 5 F and G) in the novel object recognition test. Control animals were used as a reference which showed both novelty and spatial memory at its maximum (155±5 sec). The track plot represents the correct and wrong alternation in spontaneous alternation of T-maze experiment (Fig 5H). Both SR4-02 (43.33±4.4) and SR4-04 treated group (43.33±3.3) exhibited better working memory in T-maze experiment close to 50% correct alternation rate with no significance (P=0.083) compared to the rest of the groups SR4-03 (38.33±4.4) and SR4-01 (40.33±2.8) (Fig 5I). Based on the outcomes of the all the cognitive tests conducted, SR4-02 and SR4-04 treated mice have shown a significant improvement in learning and memory functions.
Polycyclitol adjunctives alleviated cdk5/p25 based tau hyperphosphorylation
After performing the cognitive tests, we euthanized experimental animals, collected the whole brain samples and stored at -80oC. Outcomes of IHC staining show a significant decrease in phosphorylated tau at Ser396 inclusions in cortical ***p<0.001 (SR4-01; 37.67± 2.728, SR4-02; 55.33±1.764, SR4-03; 31.67± 3.383, SR4-04; 39.67± 2.728) (Fig 6 A and B) and hippocampal regions (SR4-01; 40.33±1.453, SR4-02; 29±2.082, SR4-03; 28.67±1.202, SR4-04; 37±1.528) (Fig 6 A and C) of all the treated groups compared to cortical areas with scattered tau expression (133±3.512) (Fig 6 A and B), superficial, medial entorhinal cortex of CA1 and DG in hippocampal brain regions (89.33± 5.812) in CM group (Fig 6 A and C). Overall, of all the adjunctive therapies, SR4-02 group exhibited a significant downregulation of phospho tau at Ser396 in hippocampal region; whereas SR4-03 exhibited a significant decrease of phospho tau Ser396 expression in cortical regions of CM infected animals. We found excessive deposition of tau inclusions in hilar region of CM. We also identified loss of hippocampal neuronal density especially pyramidal neurons of CA1 and CA3 regions in Golgi impregnated brain sections (Fig 7A). Dendritic arborization fulfils specialized circuit functions during cognitive processes. According to anatomical features, dendrites are categorized as distal and proximal. We observed that length of distal and proximal dendritic lengths of hippocampal neurons was significantly restored in SR4-02; distal:13.5 μm±0.41, proximal:13±0 and SR4-04 group; distal:13±0, proximal: 13±0.27 (***p<0.001) compared to rest of the treated groups (SR4-01;distal:9.5±0.139, proximal:8.0μm±0.27, SR4-03;distal:8.5μm±0.13, proximal:10μm±0.27) and CM group (distal:5.5μm±0.13, proximal: 3.5μm ±0.13) (Fig 7 B). Similarly, distal and proximal dendritic lengths of cortical neurons of SR4-02 (distal: 16.0μm ±0.13, proximal: 19.0μm ±0.27) SR4-04 (distal: 17.5μm ±0.13, proximal: 14.5μm ±0.13) (***p<0.001) was significantly arborized compared to SR4-01 (distal: 10.5μm ±0.13, proximal: 8.5±0.13) and SR4-03 groups (distal: 7.5±0.13, proximal:11.5μm ±0.13) (Fig 7 C). Western blot data showed a significant reduction in the expression of phospho tau Ser396 in SR4-03 (***p<0.001) (142.18±0.06) and SR4-04 (132.74±0.07) compared to SR4-01 (663.95±0.11) and SR4-02 (333.93±0.08) treated and CM group (2244.84±0.10). Previously our group has shown overexpression of cdk5 and phospho cdk5 at Ser159 (Czapski et al., 2016) during p25 generation in ECM (Kumar and Babu, 2020). Total form of cdk5 expression was significantly reduced in SR4-02 (0.368±0.46), SR4-03 (0.366±0.21) and SR4-04 (0.61±0.0.06) (***p<0.001) compared to CM (1.166±0.10). We observed significant reduction in expression of the activated form of cdk5; phospho Ser159 in SR4-03 (9.15±0.11) and SR4-04 (6.85±0.01) (***p<0,001) compared to SR4-01 (16.0±0.05) and SR4-02 (16.52±0.07). As expected, cdk5 was overexpressed in CM (22.82±0.01). We observed that there was no significance in cdk5 expression between SR4-01(1.037±0.05) and CON group (1.0±0.07). Excessive activation of cdk5 leads to degradation of p35 to p25. Our findings showed that p35 expression was significantly reduced in CM (***p<0.001, 0.32±0.23) and all the adjunctive treated groups show similar restoration of p35 protein (SR4-01: 1.174±0.11; SR4-02: 1.30±0.06; SR4-03: 1.191±0.15 and SR4-04: 1.279±0.10). Further, we found that the cleaved product of p25 was overexpressed in CM (2.54±0.10) and was significantly reduced in SR4-04 (0.068±0.1) (***p<0.001) compared to rest of treated groups (SR4-01: 2.438±0.11; SR4-02: 2.071±0.07; SR4-03: 0.591±0.3). The expression pattern of all the above proteins was quantified after normalization with GAPDH (Fig 8).