Heat stress modulates the GSK-3β levels and Tau phosphorylation

Alzheimer’s disease is a prominent neurological disorder, which leads to progressive dementia. The microtubule-associated protein Tau is been considered as one of the major causes of Alzheimer’s disease. Physiologically Tau assists in the stabilization of microtubules, contrary to this the pathological state of Tau results in the formation of neurotoxic tangles of Tau. The posttranslational modifications, such as GSK-3β-mediated Tau phosphorylation results in the generation of Tau pathology. Neuroinflammation generated in Alzheimer’s disease, contributes to elevated body temperature. The aim of present work is to study the effect of high temperature on Tau phosphorylation. The neuroblastoma cells were exposed to heat stress for 40 minutes. The immunofluorescence and western blot studies suggested that high temperature increases the levels of GSK-3β in cells. Heat stressed cells was also observed to have elevated levels of phosphorylated Tau. Additionally, heat stressed cells found to have modulated nuclear transport as the level of Ran was reduced. The results of present work suggested that increased temperature could be considered as a risk factor in Alzheimer’s disease as it elevated the GSK-3β levels in cells thus, resulting in increased Tau phosphorylation.


Introduction
Alzheimer's disease is the neurodegenerative disorder, resulting in symptoms-like memory loss, behavioural impairment and locomotory deficits 1, 2 . Alzheimer's disease is hallmarked by two types of protein aggregates, which are senile plaques composed of amyloid-β peptide and neurofibrillary tangles (NFTs) composed up of microtubule-associated protein Tau 3,4 . The action of gamma-secretase on amyloid precursor protein (APP) results in the generation of amyloid β-42 peptides, leading to generation of extracellular senile plaques 5 . Tau protein is considered to be a cytoskeletal protein, having role in stabilization of the microtubules [6][7][8] . In the pathological state Tau detaches from microtubules and lead to the formation of intracellular neurofibrillary tangle NFTs 9,10 . Tau is a 441 amino acid long natively unfolded protein, the domain organization of Tau comprises of projection domain and microtubule-binding domain 10,11 . The four repeat region present in microtubule-binding domain is associated with the Tau pathology 12 . The four repeat region is prone to several posttranslational modifications including phosphorylation, glycation, glycosylation etc. Tau phosphorylation is one of the major cause of pathologic state of Tau 13 . Tau phosphorylation contributes to several defects in cell signalling leading to neurotoxicity 14 . The increased load of Tau phosphorylation leads to generation intracellular ROS 15 . Heavy metal such as mercury and iron are known to modulate Tau phosphorylation 16 . Similarly, abnormal phosphorylation of Tau results in reduced microtubule binding of Tau 17 . Thus, phospho-Tau and the Tau specific kinases are considered as a prime target for therapeutic studies in AD. GSK-3β and CDK5 are the principle Ser/Thr kinases, while Src family kinases (SFKs), FYN and the ABL family kinases phosphorylate tyrosine 18 . Several compound such as leptin, which downregulates Tau phosphorylation are designated as potent molecules against AD 19 . Similarly metal ions as Lithium also showed promising results in reducing Tau phosphorylation by inhibiting GSK-3β 20 . GSK-3β and CDK5 phosphorylates Tau on various sites which ultimately results in Tau pathology 21 . Additionally, these kinases are also reported to phosphorylate the GPCR proteins, which contributes to AD pathology 22 . GSK-3β gets phosphorylated at Ser-9 leading to generation of its inactive state pGSK3-β 23 . Several studies have suggested increased levels of GSK-3β in AD condition. Fluctuation of body temperature has been observed in AD patients. Hypothermia has also been marked as a risk factor for AD. The published studies have suggested that hypothermia might down regulate the GSK-3β expression 24 . On contrary the reports suggested that as a result of neuroinflammation AD patient may suffer increased body temperature 25 .
The deregulation of nucleocytoplasmic transport is one of the consequences of neurodegeneration.
Reduced levels of Ran has been observed in case of neurodegeneration 26 . Similarly the deformities in the arrangement of nuclear pore complex (Nups) also studied in neurodegenerative disease 27 . The aim of our work is to study the effect of increased temperature on GSK-3β levels and Tau phosphorylation. The work was intended to investigate the fact weather increased body temperature could be one of risk factors for AD patients.

Heat Stress increases GSK-3β levels in cells
GSK-3β is the Ser/Thr kinase, which targets microtubule-associated protein Tau. The increased phosphorylation of Tau leads to aggregation. Thus, GSK-3βis considered to be associated with Alzheimer's disease. Neuroinflammation results in elevated body temperature in Alzheimer's disease (AD) 28 . Here we observed the effect of high temperature on GSK-3β levels in neuronal cells. Earlier studies have suggested that at temperature of 43°C the cells experiences the heat stress leading to generation of several stress responses 29 . Thus in our studies, the cells were incubated at 43°C for 40 minutes to induce the heat stress ( Figure 1A). The immunofluorescence studies suggested that heat stress increases the GSK-3βlevels in cells. The elevated GSK-3β indicated that heat stress may contribute to increased levels of Tau phosphorylation ( Figure 1B). The quantification of immunofluorescence images supported the results that intensity of GSK-3β increased after exposure to high temperature. The western blot analysis of heat stressed cells suggested that the levels of GSK-3β elevated after heat stress, whereas no changes in pGSK-3β levels were observed. Thus, the above results indicated that heat stress might elevate the levels of GSK3-β, which could be reason for induction of Tau pathology.

Heat Stress elevates phospho-Tau levels in cells
Several factors contribute to the generation of Tau pathology, among these phosphorylation of Tau is considered as leading cause of Tau pathology. Tau protein has 85 sites which are targeted by various kinases for of phosphorylation. GSK-3β, which phosphorylates Tau, at sites such as S199, S202,

The effect of high temperature on nucleocytoplasmic transport
The nucleocytoplasmic transport in cells is govern by several carrier proteins. Ras-associated nuclear protein (Ran) is the key protein for nucleocytoplasmic transport. Recent studies suggested that under neurodegenerative condition the Ran gradient gets disturbed, which results in neuronal death 30 . The objective of our study was to observe the effect of high temperature on nucleocytoplasmic transport.
( Figure 3A). The immunofluorescence images indicated that the levels of Ran were reduced in heat stressed cells. The modulation in Ran levels could be due to disturbed nuclear transport ( Figure 3B).
The quantification results suggested that the intensity of Ran lowers down in the heat stress cells ( Figure 3C). The immunoblot analysis suggested that the level of Ran protein were decreased in heat stressed cells ( Figure 3D). On contrary, we observed no modulation in NUP358 arrangement in heat stressed cells. Thus, these studies evidenced that elevated temperature could modulate the nucleocytoplasmic transport, resulting in the generation of neurodegeneration.

Discussion
The neurodegeneration includes diseases such as include Alzheimer's disease, fronto-temporal dementia, Parkinson's disease, vascular dementia, lewy body dementia, Posterior Cortical Atrophy, Creutzfeldt-Jakob Disease etc 31-33 . The protein aggregates of amyloid-β-42 and the microtubuleassociated protein Tau are considered as the major cause of Alzheimer's disease 6,13 . AD is symptomized by a spectrum of symptoms including impaired cognition, memory deprivation, emotional imbalance and problem in performing routine activities 9 . Under the pathological condition, the hyperphosphorylation of Tau leads to the generation of several structural and functional modifications resulting in neurotoxicity 34 . Cells have various classes of kinases, which include serine/threonine kinase and tyrosine kinase 34,35 . GSK-3β and CDK5 prominent Tau targeting kinases Tau 36 , which are considered to be involved in generation of Tau pathology 37 . Several studies claim the involvement of GSK-3β in AD pathology, which includes memory impairment, locomotry dysfunction and behavioural impairments 38 . The amyloid-β-induced neurotic damage have been considered to be mediated by GSK-3β activation 39 . Similarly, increased levels of GSK-3β was observe in the brain of AD patients 40 . Recent studies suggested that the silencing of GSK-3β reduces the tangles formation in mouse model of AD 41 . The selective GSK-3β inhibitor SAR502250 was reported to reduce the behavioural impairment and neuropsychiatric symptoms in rodent AD models 42 . GSK-3β is leads to apoptotic cell death by inhibiting PI3K pathways 43 . In addition to AD, GSK-3β is studied to be involved in other Tau related-frontotemporal dementia 38 . GSK3-β, also  pyrazolopyrimidine are also the compounds studied to have effect in downregulation of GSK-3β 48 .
The hypothermia in patient with neurological disorders have been reported to modulate the GSK-3β levels 24,49 . The AD patients also suffer the increased core body temperature as a result of activated inflammatory pathways 25 . The effect of high temperature on GSK-3β levels were not studied yet. Here we observed that heat stress increased the GSK-3β expression in cells, which ultimately led to  increased phospho-Tau levels in neurons. Ran is one of the key protein of nuclear transport 50 .
Phospho-Tau has also been reported to disrupt the nuclear transport by reducing the Ran gradient in cells 30 . In our study we have also monitored that heat stress results in reduction of Ran levels in cells indicating the impaired nucleocytoplasmic transport. Thus, we suggest that heat stress could act as risk factor in AD which ultimately led to Tau hyperphosphorylation.

Conclusion
Tau phosphorylation is considered as a major cause of Tau pathology. The GSK-3β is associated with Tau phosphorylation. The results of present work suggest us that high temperature could increase the expression of GSK-3β and phospho-Tau in neurons. Additionally, heat stress modulated the nuclear transport by the reducing level of Ran although the Nup358 arrangement and nuclear morphology was observed to be unaltered. Thus, the overall results suggest that high temperature could contribute to AD pathology and hence, it could be considered as a risk factor for AD.

Immunoblot assay
The mouse neuroblastoma cells Neuro2a (ATCC CCL-131) were acquired from ATCC. The cells were maintained in advanced DMEM/F-12 media supplemented with 10% fetal bovine serum. The cells were kept in incubator at 37°C supplied with 5% CO 2. The cells were regularly passaged and maintained.
The effect of heat stress were studied in mouse neuroblastoma cells (Neuro2a cells). For the western blot analysis neuronal cells were seeded at density of 1.5X10 5 cells/well in 12 well poly-lysine coated plate. The cells were subjected to heat stress at 43°C for 40 minutes. The cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail. The lysate was centrifuged at 12000 rpm for 20

Immunofluorescence
The immunofluorescence studies were further carried out to validate the effect of heat stress on various cellular signalling. Neuro2a cells (25,000 cells/well) were divided into two groups, group one comprised of cells exposed to heat stress and second group cells were kept at 37°C, which was designated as cell control. After the incubation, the cells were washed with 1X PBS, followed by 4% paraformaldehyde to fix the cells. The cells were permeabilized using 0.2% Triton X-100 followed by

Statistical analysis
The statistical data for the fluorescence measurement or viability assay was plotted by using either duplicate or triplicate reading. Untransformed (raw) data were analysed and plotted by SigmaPlot 10.0 software. The data was analysed for the significance by Student's t-test.
S.C, T.D designed and carried out the experiments. T.D and S.C analyzed the data. S.C. conceived the idea of the project, provided resources, supervised and wrote the manuscript. All authors contributed to the discussions and manuscript review.