Neuroprotective mushrooms

Introduction

Neurodegenerative diseases include, but are not limited to, Alzheimer’s and Parkinson’s diseases (AD and PD), Huntington’s disease (HD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). Neurological disorders are the leading cause of global physical and cognitive disability, and currently affect approximately 15 % of the worldwide population [1]. In addition to direct costs associated with medical management of neurodegenerative diseases, indirect costs are associated with job loss, caregivers, medical equipment, and home safety [2]. Currently, available medications are palliative. No current therapies can halt or reverse cognitive or motor dysfunctions.

While multiple natural materials have been touted to positively affect the course of disease by possessing bioactive components, there is limited to no evidence that any protects or ameliorates disease. One example is edible mushrooms that are a known source of bio-compounds that have nutritional, pharmacological, and medicinal values [3]. For example, the fruiting body and dried mycelium extract of different mushrooms contain potential therapeutic components [4]. Moreover, prior studies showed that mushrooms do have antioxidative, anti-inflammatory, immunomodulatory, antimicrobial, and anticancer properties [5–7]. Thus, the consumption of mushrooms could potentially boost immune responses or affect the signs and symptoms of disease [8]. Notably, mycelium extracts and isolated bioactive compounds (melatonin, ergosterol, terpenoids, and phenolic compounds) extracted from mushrooms have been observed to demonstrate a spectrum of ameliorating effects on AD. These include, but are not limited to, neuroprotective and anti-inflammatory activities. Mushrooms from fungi such as Grifola frondosa, Lignosus rhinocerotis, Hericium erinaceus, may positively affect cognitive functions [9]. The aqueous extract of Ganoderma lucidum (500 mg/mL) was found to recover the synaptic dysfunction in rat cortical neurons via c-Jun N-terminal kinase (JNK) and P38 kinase pathway [10]. Furthermore, microglial activation has been significantly inhibited by G. lucidum extract, and consequently, reduced the dopaminergic neuron death in PD [11]. Mushrooms’ diverse biological functions that help to prevent age-based neuronal dysfunctions have received widespread attention. Studies have begun to identify mushroom’s active compounds [9, 12, 13]. This may be only a beginning in discovery toward implementation as research continues to seek alternative remedies through mycotic medicines.

Over the past decades, proteomic studies have been developed and used to study protein content amongst a broad variety of cells, tissues, and organisms, and notably have achieved relevancy in works to discover mycotic medicines [3]. These studies were performed to identify mushroom proteins with potential as antioxidant, anti-inflammatory, anticancer, anti-diabetic, antimicrobial, and apoptotic effectors. The use of proteomics for identification of fungal homologs with relevance to human function remains a challenge due to limitations in precisely identifying human proteins and extended efforts to obtain a complete proteome [14]. In our efforts to uncover the proteomic signature of mushrooms and putative activities, we utilized label-free quantification (LFQ) mass spectrometry-based methods to identify the proteins in the fruiting bodies of both Laricifomes officinalis (agarikon) and G. frondosa (maitake). We performed functional and pathway enrichment analyses of the relevant identified proteins. The data began to unravel a spectrum of linked biological processes, molecular functions, and immune responses that point to novel neuroprotective mushroom biological activities.

Materials and methods

Results

Proteins were identified based on the human proteome database. Proteins were considered unique when expressed in one species but not the other. The proteome of agarikon included 117 unique proteins, while that of maitake included 97 proteins (Supplementary file 1). To identify the biological processes and molecular functions related to unique proteins, we performed ClueGO gene ontology (GO) which showed enrichment of beta-tubulin:GTP cofactors, cornified envelope-bound lamellar bodies, aldehyde dehydrogenase, and vascular endothelial growth factor receptor signaling pathways in agarikon (Figure 1A and Supplementary file 2). The protein repertoire unique to maitake showed enrichment of multiple signaling pathways such as phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants, KIT in disease, ERBB2, non-receptor tyrosine kinases, tyrosine-protein kinase 6 (PTK6), mitogen-activated protein kinase 1 (MAPK1)/MAPK3 (Figure 1B and Supplementary file 2).

Figure 1:

Pathway enrichment of differentially expressed unique proteins in agarikon and maitake mushrooms. Gene ontology (GO)-term functional enrichment by 5 categories (immune response, biological process, cellular component, KEGG, and Reactome) performed using Cytoscape in conjunction with the plug-in ClueGO. Enriched signaling pathways of unique proteins for (A) agarikon and (B) maitake.

Principal component analysis (PCA) of samples of both species is shown in Figure 2A. Volcano plot and heatmap depicting differentially expressed proteins in maitake compared to agarikon are shown in Figure 2B and C. Out of 134 proteins common to agarikon and maitake, 58 proteins (55 upregulated proteins and 3 downregulated proteins) were significantly changed between the two species (Supplementary file 3). To elucidate the potential protein protective activities, we performed functional and pathway enrichment analysis of maitake against agarikon using IPA. Multiple signaling pathways were found enriched, including BAG co-chaperone 2 (BAG2) (p=7.94 × 10⁻¹²), unfolded protein response (p=2.24 × 10⁻⁴), mitochondrial protein import (p=1.29 × 10⁻³), protein ubiquitination (p=7.94 × 10⁻¹¹), regulation of apoptosis (p=2.29 × 10⁻⁸), microautophagy (p=9.55 × 10⁻⁷), glycolysis I (p=1.15 × 10⁻⁶), SNAP receptor (SNARE) (p=8.13 × 10⁻⁵), and immunogenic cell death (p=1.82 × 10⁻⁴) (Figure 3 and Supplementary file 4). These results show proteins that delineate immune functions related to potential neuroprotective mechanisms such as autophagy, apoptosis, and glycolysis [17–21]. In Cytoscape ClueGo analysis, the proteins were divided into upregulated and downregulated clusters. The upregulated protein cluster showed enrichment of multiple biological and molecular functions with 50 % relationship to Parkinson’s disease (PD) and prion disease (Figure 4A and Supplementary file 5). This suggests that the proteome of agarikon and maitake share common proteins that are related to different biological functions involved in PD and other prion disease processes. In stark contrast, the downregulated protein cluster showed a dominance of Reactome reactions associated with keratin type I and type II interactions (Figure 4B and Supplementary file 5). PPIs were identified as related to five categories including biological processes, cellular components, molecular functions, KEGG pathways, and Reactome pathways, and were validated using STRING analysis for interactive networks for both upregulated and downregulated common proteins (Supplementary file 6). Complex interactions of genes involved in biological processes (such as substantia nigra development, protein refolding, and cellular response to unfolded protein); sub-cellular components (such as proteasome complex); molecular functions (such as unfolded protein binding and misfolded protein binding); and KEGG pathways of different neurodegenerative diseases (NDs) including AD, PD, prion disease, and ALS are shown in Supplementary file 6.

Figure 2:

Differential proteomic analysis of agarikon and maitake common proteins. (A) Principal component analysis (PCA) showing the three top components used for proteomic analysis. (B) Volcano plot showing the fold change plotted against the p value highlighting significantly changed proteins in maitake compared to agarikon. Red – upregulation and green – downregulation; p≤0.05 and an absolute fold change ≥2. (C) Heatmap showing hierarchical clustering of agarikon and maitake FDR-adjusted proteins. Red – upregulation and green – downregulation. Sample_A, agarikon; sample_M, maitake.

Figure 3:

Pathway enrichment of differentially expressed common proteins in agarikon and maitake mushrooms. Canonical pathway enrichment analysis of maitake proteome compared to agarikon proteome was performed using IPA (Qiagen). Orange color (activation of pathway), blue color (inhibition of pathway), and grey color (no activity pattern of pathway). The p value ≤0.05.

Figure 4:

Pathway enrichment of up- and down-regulated common proteins in agarikon and maitake mushrooms. Enriched signaling pathways of common mushroom proteins that were (A) upregulated or (B) downregulated. Gene ontology (GO)-term functional enrichment by 5 categories (immune response, biological process, cellular component, KEGG, and Reactome) performed using Cytoscape in conjunction with the plug-in ClueGO.

Discussion

Prior studies showed that natural product consumption affects NDs due to protective functions of bioactive molecules in these products [22–24]. Mushrooms possess potent medicinal properties with antioxidant, anti-inflammatory, immunomodulatory, and antimicrobial properties [9, 25]. To our knowledge, the current study is the first to uncover the proteomic signature of mushrooms and their related putative neuroprotective processes. We identified proteins in the fruiting bodies of agarikon and maitake mushrooms. Functional and pathway enrichment analyses of identified proteins in both species uncovered significant enrichment of different signaling pathways related to BAG2, unfolded protein response (UPR), mitochondrial protein import, protein ubiquitination, microautophagy, glycolysis I, SNARE, regulation of apoptosis, and immunogenic cell death. Each alone or together support prior works where polysaccharides, terpenes, phenolic compounds, and vitamins from mushrooms have been shown to potentially affect the onset and progression of NDs [13]. While decades of research have shown that natural compounds produced by living organisms can be at the forefront of new therapeutic discoveries, few have reached basic and clinical affirmations. Mushrooms are well-established to have nutritional and medicinal properties, but have not yet been studied for their potential use in disease treatments, most notably for those linked to AD. However, products extracted from mushrooms may have beneficial outcomes in AD-related processes including the inhibition of acetylcholinesterase and beta-secretase (β-secretase); and the prevention of amyloid beta (Aβ) aggregation and neurotoxicity with linked antioxidant and anti-inflammatory activities [25].

Prions are the hallmarks of degenerative diseases and include alpha-synuclein (α-syn) in PD, and Aβ and Tau in AD. Quality control of proteins plays an important role in protein homeostasis and depends on specific chaperones that regulate the balance between protein folding, assembly, disassembly, translocation, differentiation, and degradation [26]. The co-chaperone BAG2 delivers phosphorylated Tau for degradation to the proteasome in a ubiquitin-independent manner in AD models, thus playing a fundamental role in the maintenance of neuronal structure [27]. In addition, stabilization of PTEN-induced kinase 1 (PINK1) by BAG2 triggers Parkin-mediated mitophagy and protects neurons against 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress in an in vitro cell model of PD [28]. Agarikon and maitake proteomes were enriched in BAG2 pathway activity with neuroprotective functions.

Defects of mitochondria and endoplasmic reticulum (ER) were reported in different NDs [29, 30]. The main functions of the ER are synthesis and folding of proteins along the secretory pathway, a process mediated by luminal resident chaperones and foldases. The UPR provides a coordinated response that is initiated in the ER and allows the correct folding of proteins [20]. Alterations in ER functions, such as altered calcium levels, increase oxidative stress and/or dysfunction of protein N-glycosylation causing the accumulation of misfolded proteins in the ER, which triggers the ER stress that activates the UPR to augment the ER folding capacity [31]. Two major degradation pathways, the ubiquitin-proteasome system and the autophagy-lysosomal pathway, remove damaged or misfolded proteins to prevent accumulation and maintain homeostasis of cellular proteins [32]. Aging, a primary risk factor of many NDs, is associated with a general reduction in proteasomal degradation and autophagy with the consequent accumulation of potentially neurotoxic protein aggregates of Aβ, Tau, and α-syn [32]. In concert with proteasomal degradation, ubiquitination is associated with degradation signals to process aberrant proteins which form aggregates and lead to neuronal cell damage and death if not chaperoned to the proteasome. Furthermore, autophagy is one of the major intracellular mechanisms to eliminate misfolded proteins and maintain proteostasis [33]. Dysregulated autophagy is increasingly considered to play key roles in most NDs, thus the regulation of autophagy is proposed as a potential therapeutic avenue for these diseases [34, 35]. Coincidentally, agarikon and maitake proteome exhibit proteome pathways that are enriched for protein homeostasis and autophagolysosomal activities.

SNARE proteins are small proteins of around 100–300 amino acids that mediate the fusion of biological membranes by localizing both at the vesicular membrane and on the target membrane [36]. SNARE proteins have fundamental roles in neuronal functions such as neurite initiation and outgrowth, axon specification, axon extension, synaptogenesis, and synaptic transmission [37]. The formation of the SNARE complex was found to be fundamental for vesicle fusion, vesicle recycling, and neurotransmitter release [38]. Inhibition of the formation of the SNARE complex, defects in the SNARE-dependent exocytosis, and altered regulation of SNARE-mediated vesicle fusion were found to be associated with neurodegeneration [39–41]. Agarikon and maitake protein-enriched SNARE signaling pathways support mushroom neuroprotective functions.

The brain depends mainly on glucose as a source of energy to maintain normal functions. Aging impairs cerebral glucose metabolism, reduces mitochondrial biogenesis, and decreases ATP levels [42]. Glycolysis and mitochondrial functions are dysregulated in individuals with NDs [43–45]. Decreased glycolytic flux has been shown to correlate with the severity of amyloid and Tau pathology in AD patients [46, 47]. Glycolytic dysfunction has also been observed in PD [18], HD [48], and ALS [49], thus identifying glycolytic dysfunction as a common pathway contributing to neurodegeneration. In general, glycolysis upregulation was found to be neuroprotective in several different NDs [50, 51]. In that vein, agarikon and maitake protein repertoires showed that upregulation of glycolysis offered another possible neuroprotective role.

Immunogenic cell death plays an important role in immunosurveillance by which immune cells look for and recognize foreign pathogens such as bacteria, viruses, or pre-cancerous and cancerous cells in the body [19]. NDs are characterized by progressive loss of particular populations of neurons whereby programmed cell death (apoptosis) has been largely implicated in their pathobiology [52]. The homeostatic maintenance of most organs and tissues is ensured by a controlled balance of cell proliferation and elimination via apoptosis throughout the life of an organism. However, the nervous system is different from most organs because neuronal cell division and proliferation are primarily restricted to the embryonic development stage. The threshold required to induce apoptosis becomes much greater once the mature nervous system is established, resulting in markedly less neuronal cell death. Indeed, mature neurons strikingly restrict the apoptosis pathway after development to ensure long-term survival and maintenance throughout life [53]. The protein repertoires of agarikon and maitake comprised proteins linked regulation of immunogenic cell death and apoptosis which have been shown to serve protective functions for neurons and the entire nervous system. Altogether, with the evaluation of common and unique proteins amongst the agarikon and maitake proteomes, we uncovered signaling pathways and molecular functions that are related to neuroprotective activities in the realm of human signaling pathways. To our knowledge, this study is the first to unravel the mushroom proteome of fungal species and expose proteins involved in mechanisms associated with neuroprotective activities for various NDs.