Phosphoproteomic analysis reveals Akt isoform-specific regulation of cytoskeleton proteins in human temporal lobe epilepsy with hippocampal sclerosis

https://doi.org/10.1016/j.neuint.2019.104654Get rights and content

Highlights

  • Differential activation of Akt isoforms was observed across different hippocampal tissues samples from TLE patients.

  • Akt isoforms plays an essential role in hippocampal sclerosis.

  • Cytoskeletal protein phosphorylation events plays a major role in hippocampal sclerosis.

Abstract

Akt is one of the most important downstream effectors of phosphatidylinositol 3-kinase/mTOR pathway. Hyperactivation and expression of this pathway are seen in a variety of neurological disorders including human temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). Nevertheless, the expression and activation profiles of the Akt isoforms, Akt1, Akt2, and Akt3 and their functional roles in human TLE-HS have not been studied. We examined the protein expression and activation (phosphorylation) patterns of Akt and its isoforms in human hippocampal tissue from TLE and non-TLE patients. A phosphoproteomic approach followed by interactome analysis of each Akt isoform was used to understand protein-protein interactions and their role in TLE-HS pathology. Our results demonstrated activation of the Akt/mTOR pathway as well as activation of Akt downstream substrates like GSK3β, mTOR, and S6 in TLE-HS samples. Akt1 isoform levels were significantly increased in the TLE-HS samples as compared to the non-TLE samples. Most importantly, different isoforms were activated in different TLE-HS samples, Akt2 was activated in three samples, Akt2 and Akt1 were simultaneously activated in one sample and Akt3 was activated in two samples. Our phosphoproteomic screen across six TLE-HS samples identified 183 proteins phosphorylated by Akt isoforms, 29 of these proteins belong to cytoskeletal modification. Also, we were able to identify proteins of several other classes involved in glycolysis, neuronal development, protein folding and excitatory amino acid transport functions as Akt substrates. Taken together, our data offer clues to understand the role of Akt and its isoforms in underlying the pathology of TLE-HS and further, modulation of Akt/mTOR pathway using Akt isoforms specific inhibitors may offer a new therapeutic window for treatment of human TLE-HS.

Introduction

Temporal lobe epilepsy with hippocampal sclerosis (TLE-HS) is one of the most common and refractory focal epilepsy syndromes that usually starts in childhood (Berg et al., 1999; Pitkanen and Sutula, 2002). Hippocampal sclerosis accounts for at least 80% of all temporal lobe seizures (Tatum, 2012; Vezzani and Friedman, 2011). Neuronal loss, axonal sprouting and synaptic reorganization of hippocampal structures are the main pathological changes that occur in chronic TLE and form the pathological basis of refractory epilepsy (Sutula, 2002). Previous studies confirmed several trends in resected human hippocampal tissues from TLE patients that unquestionably contributed to a higher understanding of development and progression of TLE. Resected hippocampal tissues from patients and animal models of this disorder revealed different histopathological changes including the neuronal loss in the CA1 and CA3 regions, sclerotic gliosis, and aberrant mossy fiber sprouting from dentate granule cells (Bae et al., 2010; Cascino, 2005; De Lanerolle et al., 2003; Epsztein, 2005; Melø et al., 2010; Sharma et al., 2007; Wieser and Epilepsy, 2004; Yang et al., 2010). Regardless of thorough studies, details of modifications at the molecular level responsible for the abnormal histological changes observed in TLE-HS patients are principally unknown. Further, understanding of these molecular changes may provide clues for better assessment of disease pathology that may enable us to identify novel therapeutic options for the treatment of TLE-HS.

PI3K/Akt/mTOR is an evolutionarily conserved signaling pathway that plays an essential role in controlling many physiological and pathological processes. Akt (also known as Protein Kinase B–PKB) is an extensively studied protein that belongs to the AGC family of serine/threonine kinases. Akt is not only known for its ubiquitous distribution but also for its critical roles in nervous system development (Crowder and Freeman, 2000; Dudek, 1997; Ohba et al., 2004). Akt is revealed to be involved in numerous biological processes and pathologies, such as metabolic regulation, cell growth, survival, proliferation, cancer and neurodegenerative disorders (Chen et al., 2003; Emamian et al., 2004; Kwon et al., 2006; Liao and Xu, 2009; Warby et al., 2009; Zala et al., 2008). Recent studies have shown that Akt plays a significant role in epileptogenesis in TLE. Studies have shown constitutive activation of Akt and its downstream signaling molecules in in vivo and in vitro models of epilepsy (Wei et al., 2018; Zhu et al., 2017). Furthermore, human hippocampal tissue samples from TLE-HS patients have shown deregulated Akt/mTOR signaling pathways further proving evidence of pathological role(s) mediated by this pathway in (Talos et al., 2018; Xiao et al., 2015).

The Akt family of proteins in mammalian cells exists at least in three isoforms, encoded by separate gene loci: Akt1, Akt2 and Akt3. Akt1 is distributed ubiquitously, whereas Akt2 is primarily expressed in the liver, skeletal muscle and adipose tissues; and Akt3 is mostly seen in the brain and testis (Dummler and Hemmings, 2007). Functional roles of Akt isoforms were identified by targeted disruption using specific knockout mice models. Akt1 knockout mice displayed placental hypotrophy and a reduction in total body weight, while Akt2 knockout mice demonstrated a diabetes-like syndrome with mild growth retardation. Akt3 knockout mice showed a 25% decrease in brain size compared with wild-type brains with no difference in body size suggesting that Akt3 plays a crucial role in postnatal brain development (Cho, 2001; Cho et al., 2001; Garofalo et al., 2003; Yang et al., 2003). Diez et al. (2012)elucidated the roles of Akt isoforms in neuron viability and axonal growth. Also, an investigation from Levenga et al. (2017) revealed that the ratio and timing of expression of Akt isoforms are different during the development of cortical and hippocampal neurons along with differential regulation of the isoform specific downstream signaling pathways during nervous system development.

Akt, a crucial signaling molecule in the PI3K/mTOR pathway cross-talks with many other signaling pathways involved in crucial cellular functions. When a dysregulated Akt pathway is targeted with Akt inhibitors, all three isoforms are blocked and may present unwanted side effects. Therefore critical analysis of the roles of specific Akt isoforms in pathological disorders and further development of specific molecular modulators are necessary to address this problem. However, comparative studies on the endogenous expression and activity profiles of Akt isoforms in neuronal cells during diseased brain pathology are largely missing. Hence, for the first time we analyzed Akt isoform expressions and activity profiles in hippocampal tissue samples from TLE-HS patients and further identified different downstream targets phosphorylated by Akt isoforms using the phosphoproteomic approach.

Section snippets

Human subjects

The TLE-HS (Test; temporal lobe sclerosis) and non-TLE (Controls; no sclerosis; Rasmussen's encephalitis, West syndrome and focal cortical dysplasia), patients planned for epilepsy surgery were enrolled in the study (Suppl. Table 1). The control tissue used for this study is superior to autopsy material (used in other studies) because the control tissue has been resected in the same manner as tissue from TLE-HS patients. Moreover, the control and TLE tissues were subjected to similar regimens

Clinical and pathological characterization of study samples

The mean age at surgery in the TLE-HS and non-TLE groups (Table .1) were 14.5 and 10.3 years respectively with an average age at seizure onset of 6.42 and 4.42 years respectively. The mean duration of epilepsy was 9.63 and 6.63 years in the two groups. Predominant seizure types were medically intractable with focal onset. One patient had status epilepticus as one of the clinical symptoms. All TLE-HS patients were on polytherapy with two or more antiepileptic drugs. All the non-TLE patients were

Discussion

The most common pathological finding in temporal lobe epilepsy (TLE) patients undergoing surgery for drug-resistant seizures is hippocampal sclerosis (HS). Although many studies have indicated a strong association between hippocampal sclerosis and TLE, it remains unclear whether ongoing seizures worsen the hippocampal sclerosis. Experimental studies with epileptic animal models showed that neuronal loss in HS was due to apoptosis (Lee et al., 2001; Shinoda et al., 2004; Xu et al., 2007) while

Conclusion

A number of biochemical aspects related to the pathophysiology of TLE-HS are poorly understood. Among them are the signaling mechanisms and the complex interactions involved in the progression of TLE-HS. Herein, we attempted to understand the role(s) of activated Akt and its isoforms in the human TLE hippocampus using a phosphoproteomic approach. It appears that all three Akt isoforms are involved in cytoskeleton regulation and Akt1 and Akt2 together appear to control many pathological

CRediT authorship contribution statement

Rajesh Ramanna Valmiki: Conceptualization, Supervision, Project administration, Funding acquisition, Writing - original draft, Resources, Software, Validation, Visualization. Subhashini Venkatesalu: Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization. Ari George Chacko: Conceptualization, Writing - review & editing. Krishna Prabhu: Conceptualization, Writing - review & editing. Maya Mary Thomas: Conceptualization, Writing - review &

Acknowledgement

The study was funded by Department of Science and Technology (DST-SERB) through grants ECR/2016/001445 (Early Career Research award to RV) and Christian Medical College- Fluid Research grant. We also acknowledge the immense help of Mr Benjamin and Mr Jayadeepan in collecting hippocampus tissue samples.

Glossary

ACTB
Actin, cytoplasmic 1
ACTG1
Actin, cytoplasmic 2
ALB
Serum albumin
ATP6V1A
ATP synthase subunit alpha, mitochondrial
CaMKIIA
Calcium/calmodulin-dependent protein kinase type II subunit alpha
CCT3
T-complex protein 1 subunit gamma
CCT7
Isoform 2 of T-complex protein 1 subunit eta
CFL
Cofilin
CNP
2′,3′-Cyclic Nucleotide 3′ Phosphodiesterase
DPYSL2
Dihydropyrimidinase-related protein 2
EEF1A1
Elongation factor 1-alpha 1
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
GFAP
Glial fibrillary acidic protein
GLUD1
Isoform 2

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