3.1 Differential expression of the significantly regulated proteins
Differential expression of the proteins involved in neurotransmission, synaptic plasticity, neurogenesis, memory and learning related proteins such as neurochondrin, synaptophysin, synapsin-1, synapsin-2, synaptogyrin 3 (Syngr3), 4-aminobutyrate aminotransferase and 14-3-3 protein gamma were observed in the different rat groups (C, A and AE) (Table 1). Associative learning and long-term memory related proteins glutathione-S-transferase 3 and tenascin R were up regulated. Synaptic plasticity promoting Ras related protein Rab 5a and nerve growth factor (NGF) signaling Rap-1A were also among the significantly up-regulated group. Similar was the case for the heat shock proteins (HSP) involved in the regulation of neuronal migration (HSP 90-alpha) and apoptosis (HSp 60). Up-regulation was also observed for the proteins involved in post-synaptic excitatory potential (serine/threonine-protein phosphatase, syntaxin 1B), pre- and post-synaptic density (isoform 2 of clathrin coat assembly protein AP180) and tyrosine phosphorylation (hemopexin) in the AD compared to the mushroom-treated (AE) group.
Proteins involved in synaptic organization (neurofascin) and synaptic vesicle budding (ADP-ribosylation factor 1), vesicle mediated transport (syntaxin 1A), neuronal differentiation and development (Dihydropyrimidinase-related protein 1 and 2), axonogenesis (2', 3'-cyclic-nucleotide 3'-phosphodiesterase), axonal choice point recognition (neuromodulin) and axonal transport (neurofilament light polypeptide) were also differentially expressed in the hippocampus of the three rat groups. Beta-soluble NSF attachment protein (SNAP-β) involved in the regulation of glutamatergic synaptic transmission, disassemble of SNARE complex and synaptic vesicle priming was also up regulated. In addition to these, differentially up regulated expression of glial fibrillary acidic protein (GFAP) was also observed. GFAP is involved in long-term synaptic potentiation, neurotransmitter uptake, neurogenesis, glial and Schwan cell proliferation.
In the present study, down-regulated expression of memory and learning related proteins in the AD group was observed when compared with both C and AE group. However, there was inter-group variation in the extent of fold change in case of the down-regulated proteins. In the AD versus control group, down regulated expression of the proteins involved in dopamine decarboxylation, clusterin (stimulator of Aβ and NFT), neuromodulin, neurofascin and NCAM 1 was observed. In the AD versus AE groups, α-synuclein, synaptogyrin 1 and transgelin-3 were among the most important down regulated proteins. In addition to these, neuromodulin, excitatory amino acid transporter and park 7 were the mostly up regulated proteins in the AE versus C groups (Table 1).
Following are the AD related proteins differentially expressed in the present study
Syntaxin-1A regulates vesicular trafficking during exocytosis and trans-membranal protein insertion . Decreased expression of syntaxin-1 A in the AD rats might have affected synaptic functions . Mushroom treatment might have synaptic function improving effect as up-regulated expression of this protein has been observed in the mushroom-treated group.
Synaptogyrin-1 is involved in maintaining short- and long-term synaptic plasticity. Level of hippocampal syntaxin-1 A and synaptogyrin-1 had been found to be reduced in line with AD progression . Its lowered expression in the AD and increased level in the AE rats reveals the ameliorating effect of G. lucidum.
Neuromodulin is a neuronal growth and neurite forming protein whose level decreases in AD brains. As an CSF biomarker, its lowered level has been found in other studies also [25–27]. However, in the present study, mushroom treatment (AE) has been found to increase the abundancy of this protein as is evidenced by the up-regulated expression.
Neural cell adhesion molecule (NCAM)
NCAM plays important role in brain development and increased level of NCAM 1 in transgenic AD mouse model (Tg2576) and of NCAM 2 in human AD patients have been reported [28–29]. Current findings were compatible with the previous ones as G. lucidum treatment helped increase the abundancy of NCAM.
Endophilin A1 is a membrane bending protein involved in CNS development, apoptosis, signal transduction and microtubule based movement. AD rats’ hippocampi showed decreased expression while the control and AE rats experienced increased expression of endophilin A1 in the present study. In the temporal neocortex of the AD patients, decreased level of endophilin A1 has been observed .
Clathrin group of proteins are involved in neuronal secretory functions and synaptic maintenance . AD pathogenesis involves altered clathrin-associated membrane trafficking resulting in neurodegeneration . Between the light and the heavy chains of clathrin, impaired distribution of the former has been linked with the AD pathogenesis [31–32]. Similar pattern was observed for the AD rats in the current experiment and an increasing trend following G. lucidum treatment (AE).
Septins are GTP-binding proteins found to be co-localized with the NFT in the AD brains . Differential expressions of septins have been observed in the present study. Contrary to the findings of Musunury et al. (2014)  and Shin et al. (2004) , down-regulated expression of septin-2 had been observed in the AD versus AE group of the present study. As septin-2 is involved in synaptic plasticity, its down-regulation in the AD versus AE group bears supports to the synaptic dysfunction associated with the AD pathogenesis. However, its down-regulated expression in the CE versus AE group is of intriguing. Interestingly, the isoform-2 of septin-5 had also been found down-regulated in both the AD versus AE and AE versus C groups which is compatible with the findings of Musunury et al. (2014) , who found similar expression status in the temporal brain neo-cortex of the AD patients. Thus, differential expression even of the different isoforms of the same protein might be implicated in the AD pathogenesis and corresponding modulation demands differential therapeutic strategy. Current observation of the G. lucidum HWE upon differential expression of different isoforms of septin is a unique finding that demands further studies.
Ubiquitin carboxyl-terminal hydrolase L1 (UCH L1) is an important enzyme for maintenance of cognitive and synaptic functions . Conflicting information regarding its expression has been documented in different AD cases. Though most of the researchers have noticed decreased and oxidatively modified form of UCH L1 in AD subjects [36–38], Sultana et al (2007), reported its 1.31-fold increase in the AD brain hippocampi . Increased expression of UCH L1 was observed in the G. lucidum-treated (AE) group.
Soluble NSF-attachment protein beta (SNAP-β)
N-ethylmaleimide sensitive fusion proteins (NSF) are the part of APP and overexpressed in AD . Soluble NSF-attachment proteins are involved in intracellular membrane fusion and vesicular trafficking. Among α-, β- and γ- SNAPs, α - and γ- SNAPs are expressed in different tissues while the β-SNAP is brain specific. In AD brain, differential expression and oxidized form of SNAP-β had been detected through redox proteomics .
Neuropolypeptide h3 is a cholinergic neuro-stimulating peptide that falls in the phosphatidyloethanolamine binding protein group and is also known as Raf-kinase inhibitor protein (RKIP) and/or hippocampal cholinergic neurostimulating peptide (HCNP) . Our finding of down-regulated neuropolypeptide h3 is in agreement with those of Butterfield (2004) . Oxidatively modified loss of function of neuropolypeptide h3 impairs phospholipid asymmetry that might be involved in extrusion of phosphatidyl serine to the outer membrane of neuron and signal for apoptosis and cause neuronal death . Also, neuropolypeptide h3 mediated stimulation of acetylcholine esterase (AchE) becomes compromised and this effect is heightened when HNE interacts with AChE in presence of Aβ (1–42) in synaptosome [44–45]. Thus, in AD brains, neuropolypeptide h3 is linked with cholinergic abnormalities and altered lipid metabolism that are the early events in AD pathogenesis .
AD rats showed increased expression of annexin in the hippocampi. Previous studies have linked increased plasma annexin5 with increased AD risk . Transgenic AD mice (Tg2576) also expresses increased annexin in the brain cortex .
Glycogen synthase kinase 3 β (GSK3β)
Glycogen synthase kinase 3 β (GSK3β) is a serine/threonine kinase having diversified regulatory functions ranging from glycogen metabolism to gene transcription. Overactivity of GSK-3β has been linked with elevated Aβ production, tau hyperphosphorylation and impaired memory and learning activities . Memory affecting mechanism of GSK-3β involves interruption of intra-neuronal anterograde mitochondrial transportation and causation of “mitochondrial traffic jam” [50–51].
Serine/Threonine protein phosphatase
Serine/Threonine protein phosphatase negatively regulate memory and learning abilities by impairing synaptic plasticity and LTP . Up-regulation of serine/threonine protein phosphatase in the AD rats might contribute towards impaired memory and learning performance in the present study.
Serine protease inhibitors (serpins)
Serine protease inhibitors (serpins) regulate proteolytic processing of proteins. Previous studies indicated their increased level in plasma and CSF of AD patients . We also observed increased expression of serpins (α1-antitrypsin) in the AD rats’ hippocampi. Alpha 1-antitrypsin (A1AT) has been reported to be co-localizing with Aβ plaques and NFTs .
4.2 PPI network of the upregulated protein clusters in the AD vs AE group
In the AD versus AE group, the most notable protein networks included those involved in the biological processes such as redox mechanisms, neurogenesis, neurotransmission, neuronal development and metabolism (Fig. 1). Interacted protein networks involved in molecular functions such as SNARE binding, syntaxin binding, syntaxin-1 binding were also enriched (Fig. 1).
The main network of the upregulated proteins can be divided into several subsets with respect to their gene content such as sub-set 1 consisting of synaptogamin-1, syntaxin 1A, syntaxin 1B, beta soluble NSF attachment protein (Syt1, Stx1a, Stx1b and Napb), sub-set 2 involving AP2 forming proteins and clathrin heavy chain (Ap2a2, Ap2a1, Cltc), sub-set 3 consisting of metabolic enzymes NADH dehydrogenase, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, pyruvate dehydrogenase E1 component subunit beta and isocitrate dehydrogenase (Ndufv2, Ndufa9, Dlat, Pdhb and Idh3a) and those involved in signal transduction, membrane trafficking and cytoskeletal maintenance (Ubb, Vcp, C3, Rab7a, Rab11b, Gpi and HK1) (Fig. 1). Besides the main network, two other protein-protein interaction networks involved immune-regulatory proteins (Igg-2a, Igh-1a, IgkC and Crp) and proteins associated with Ca2+-mediated signaling and metabolism (Calm1, Camk2a, Camk2b and Ppp3ca) (Fig. 1).