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).