Discussion
Tau pathology is reported to be a biomarker for cognitive impairment involving cerebral ischemia and neuroinflammation in the early and late stages of AD (Ballatore et al., 2007; Freude et al., 2005; Iqbal et al., 2005; Kim et al., 2009; Mandelkow et al., 1996). Children less than five years old infected with CM exhibited axonal injury caused by increased plasma tau levels resulting in cognitive impairment and severe mortality (Datta et al., 2021; Medana et al., 2007). A recent study by Oscar B et al. states that dysregulation of tau protein contributes to cerebral vasculopathy and neuronal cell injury impairing cognitive outcomes during CM (Akide Ndunge et al., 2023). According to the Minxian Dai et al., aberrant phosphorylation of tau increased the disease severity by inducing conformational changes in neuronal proteins in experimental models of CM (Dai et al., 2012b). C57BL/6 mice infected withPlasmodium berghei ANKA is widely known to reiterate the symptoms of human cerebral malaria. Despite several transgenic models for tau, ECM pathology globally activates tau without transgenic tau approach. Tau hyperphosphorylation is propelled by several kinases including cdk5, glycogen synthase kinase 3 (GSK3β) and mitogen-activated protein kinase (MAPK) (Hatch et al., 2017). Previously, one of our recent studies showed a dysregulation of cdk5 signaling, imbalance between kinases (Ca2+/calmodulin-dependent protein kinase II alpha, CaMKIIα, protein kinase A, cdk5) and phosphatases (protein phosphatase-1 gamma subunit 1, PP-1γ1) in ECM. Based on our previous studies and several reports on vasculopathy based tau hyperphosphorylation, we selected the animal model for CM as an effective model for this study.
Sequestration of infected RBCs cause microvasculature damage inducing chronic hypoxia mediated elevation of VEGF (Vascular Endothelial Growth Factor) which results in expression of calcium-dependent proteases, calpain (Rénia et al., 2012). Chronic hypoxic conditions in the brain are detected in the form of elevated Hypoxia-inducible factor (HIF)-1α levels in terminally ill CM animals and human cerebral malaria (Hempel et al., 2014). Low oxygen levels trigger activation of calpain and several kinases such as mitogen-activated protein kinases (MAPKs) and extracellular signal regulated kinases (ERK1/2) activating brain-derived neurotrophic factor (BDNF) (Gao et al., 2013; Raz et al., 2019; Terraneo and Samaja, 2017). Finally, all the above conditions lead to the imbalance of kinase and phosphatases resulting in tau phosphorylation. We assume that altered vasculature, reduced blood flow and chronic hypoxia upregulate cdk5, one of the key factors in phosphorylating tau at several sites such as Ser202, Ser396/404, Thr181and Thr231 (Noble et al., 2003) in CM. Tau phosphorylation at Ser396 is considered as the earliest event in Alzheimer’s disease in which cdk5 has been implicated as potential candidate kinase (Mondragón-Rodríguez et al., 2014; Noble et al., 2003). Similarly, cdk5/p25 signaling is detected early in neurodegenerative diseases like Parkinson’s (Ao et al., 2022), amylotrophic lateral sclerosis (ALS) (Bk et al., 2019), brain injuries like subarachnoid haemorrhage (Ding et al., 2022), anxiety, depression (Takahashi et al., 2022), learning disabilities involving cognitive impairment (Kamiki et al., 2018) and cancers like glioblastoma (GBM) (Peyressatre et al., 2020). Several research studies have shown that inhibition of hyperactivated cdk5 based signaling improved hippocampal neurogenesis and restored cognitive functions in radiation induced cognitive dysfunction (Zhang et al., 2021). Cdk5 inhibition alleviated diabetic neurotoxicity related cognitive deficits in diabetes mellitus animal model (Liu et al., 2019). Drugs such as rolipram (Wachtel, 1983), cilostazol (Schaler and Myeku, 2018), sildenafil (Sanders, 2020), CM-414 (Cuadrado-Tejedor et al., 2017) reduce tau hyperphosphorylation and increased dendritic spine density in hippocampal neurons improving cognition in animal models. Interestingly, recent reports state that D-pinitol, a naturally occurring inositol alleviate hyperactivation of p25/cdk5 activity by regulating cyclin‐dependent kinase 5 regulatory subunit 1 (CDK5R1) and GSK-3 beta (tau phosphorylation regulating gene) leading to tau dephosphorylation (Medina-Vera et al., 2022). According to Jeremy Koppel et al., increased extracellular dopamine levels hyperphosphorylate tau and blocking dopamine D2 receptor results in robust decrease in tau phosphorylation (Koppel et al., 2019). Dopaminergic receptors can modulate dopamine and cyclic adenosine monophosphate regulated phospho protein (DARPP-32), which acts as molecular switch to activate or inhibit kinases (cAMP-dependent protein kinase) by activating protein phosphatase-1. We assume that there is a current necessity to identify the earliest signaling mechanism that trigger tau hyperphosphorylation and drugs that restore the balance between kinases and phosphatases in tauopathies. Our study does not determine phosphatase levels but further research has to be performed whether polycyclitols restore balance between kinase and phosphatase levels in ECM.
Till date, there has been no study conducted on the effect of polycyclitols on cognition and behavior. To our knowledge, our findings represent the first evidence describing the role of polycyclitol derivatives on learning and memory functions in ECM. Christopher D Morrone et al., reported that administration of scyllo -inositol reduced the levels of amyloid beta and reversed the cognitive decline in TgF344-AD rats (Morrone et al., 2020). However, failure ofscyllo -inositol (6 ), in phase-II trials resulted in a setback to this molecule which otherwise exhibited significant Aβ -42 lowering in patient brains. This failure was attributed to its toxicity and non-specific binding with the target protein possibly because of its small size. Considering this failure of scylloinositol (6 ) against Aβ , it was an opportunity to evaluate our recently accessed polycyclitols against the other important concerned protein ‘tau’ and map their potential in controlling its hyperphosphorylation (Rashid et al., 2020). Due to the bulkier size of polycyclitols (15 , 16-18 ), we expect them to have increased number of interacting sites which might result in a better therapeutic potential for neurodegenerative diseases involving tauopathy. According to a recent reports, tau based therapeutics could play a potential role in reversing the neuronal cell damage and cognitive impairment in ECM (Akide Ndunge et al., 2023; Dai et al., 2012b). ARM alone impairs blood-brain barrier in experimental models of CM despite improving survivability by parasite clearance (Brejt and Golightly, 2019; Dai et al., 2012a; Gul et al., 2021). In agreement to previous research studies, our group showed ARM therapy alone is unable to restore cognition (Kumar and Babu, 2022). ARM monotherapy also holds the current problem of anti-malarial resistance in patients with severe malaria. Therefore, we administered our adjuncts (SR4-01 to 04) along with ARM instead of ARM alone in our study.
RMCBS scores show that rescue therapy successfully alleviated neurobehavioral impairment after few days of administration. We strongly assume that the restoration of RMCBS behavioral parameters is solely due to the ARM as is the first line therapy for CM clearing parasites in the blood. According to Clemmer et al., survivability of ARM treated animals is near to 50 percent (Clemmer et al., 2011), and the same was observed after rescue ARM adjunctive therapy on day 30. Based on outcomes of Barnes maze experiment, we assume that SR4-02 (15 ) and SR4-04 (18 ) treated animals restored the spatial reference memory with minimal error rates. The same experimental groups exhibited a retrieval of working memory in spontaneous alternation method of T-maze experiment. SR4-02 group also showed improved learning and novelty in novel-object recognition test which was similar to the control group. SR4-01 (16 ) and SR4-03 (17 ) groups showed increased side preference while performing T-maze experiment. The increase in error rates in SR4-01 could be due to loss of neuronal density in hippocampal regions and expression of lower levels of phospho tau, p25 inducing neurotoxic stimuli. SR4-03 group exhibited improved neuronal arborization pattern but cognitive impairment still persisted. SR4-03 treated brain sections show improved neuronal arborization with lower restoration of cognition. One of the reasons could be that SR4-01 and SR4-03 contain a carbonate functionality in common compared to SR4-02 and SR4-04, which makes the former compounds highly reactive and accelerate ROS generation (Juan et al., 2021)(Sparrow et al., 2003). We assume that both SR4-01 and SR4-03 drugs with ARM interacts with hemoglobin, producing toxic oxygen free radicals; the other reason could be the non-compliance of dosage standardization.
Under pathological conditions, hyperphosphorylated tau localized to somatodendritic compartments impair neuronal arborization by dysregulating microtubule regulatory proteins, affecting microtubule stability and resulting in loss of neuronal density (LaPointe et al., 2009)(Kimura et al., 2014). Neuronal loss due to tau hyperphosphorylation is observed in hippocampal, cortical pyramidal neurons and striatal neurons in Alzheimer’s, Parkinson’s (Kaul et al., 2011) and dementia with Lewy bodies (Hatch et al., 2017). Previously, our group showed extensive neurodegeneration associated with upregulation of NADPH oxidase 2 in CA1 and dentate gyrus regions of hippocampus (Kumar and Babu, 2022) also known as early vulnerable regions of neuronal damage in neurodegenerative diseases (Davolio and Greenamyre, 1995; Planche et al., 2018). Hyperphosphorylation of tau dissociates microtubules affecting the distal ends followed by proximal ends of the dendrites of pyramidal neurons in AD (Regalado-Reyes et al., 2020). As per the findings of Golgi-cox staining, we assume that decreased lengths of distal and proximal neurons of cortical and hippocampus is a key indicator for the loss of neuronal plasticity in CM. p25 and cdk5 hyperactivation followed by tau pathology could deteriorate dendritic arborization pattern of the pyramidal hippocampal and cortical neurons in ECM. We assume that hyperphosphorylated form of tau and accumulation of p25 could be a potential reason for loss of neuronal density in CM. Restoration of neuronal density after administration of adjunctives SR4-02, SR4-03 and SR4-04 could be due to significant reduction of phosphorylated tau, p25 neurotoxicity and total cdk5 levels after ECM.