The Neuroprotective Potential of Endophytic Fungi and Proposed Molecular Mechanism: A Current Update

Millions of people are affected by neuronal disorders that are emerging as a principal cause of death after cancer. Alzheimer's disease, ataxia, Parkinson's disease, multiple system atrophy, and autism comprise the most common ones, being accompanied by loss of cognitive power, impaired balance, and movement. In past decades, natural polyphenols obtained from different sources including bacteria, fungi, and plants have been utilized in the traditional system of medicine for the treatment of several ailments. Endophytes are one such natural producer of secondary metabolites, namely, polyphenols, which exhibit strong abilities to assist in the management of such affections, through modifying multiple therapeutic targets and weaken their complex physiology. Limited research has been conducted in detail on bioactive compounds present in the endophytic fungi and their neuroprotective effects. Therefore, this review aims to provide an update on scientific evidences related to the pharmacological and clinical potential along with proposed molecular mechanism of action of endophytes for neuronal protection.


Introduction
Neurodegenerative diseases (ND) include debilitating conditions that pose a serious threat to the human health leading to progressive degeneration of nerve cells. e brain disorders like Alzheimer's disease (AD), ataxia, Parkinson's disease (PD), multiple system atrophy, autism, are significantly linked to insufficient production of neurotransmitters, abnormal ubiquitination, aggregation of abnormal proteins followed by inflammation, and oxidative stress in the central nervous system (CNS) [1][2][3]. Although, there has been progression in our understanding about ND, the potential triggers of such disorders and their molecular mechanisms are still uncertain [1,3]. Currently, no reliable cure is being available for the treatment of ND due to limited regeneration ability of CNS [4,5]. e commercially available therapies are generally symptomatic and are recommended to alleviate the manifestation of disease and also to improve the health status of the patient's life. Furthermore, the treatment includes synthetic neuro medicines, associated with severe side effects [1,6,7]. A plethora of evidence has indicated promising therapeutic potential of natural bioactive compounds including various classes like phenols, flavonoids, alkaloids, and terpenoids, with high antioxidant activity against ND [8][9][10][11][12][13].
Endophytes are an endosymbiotic class of microorganisms, majorly comprising of bacteria and fungi colonizing in the tissues of healthy plants without posing any detrimental effect to their host. ey are the treasure house of secondary metabolites such has flavonoids, alkaloids, polyphenols, saponins, and tannins with multiple therapeutic benefits [14][15][16][17]. eir richness in bioactive compounds make them fruitful candidates for drug development against different disorders, such as cancer, diabetes, hypertension, cardiovascular, gastrointestinal, and ND [18][19][20][21][22][23]. Among bacterial endophytes, there are more than 200 genera of bacterial species including Streptomyces, Agrobacterium, Acinetobacter, Bacillus, Pseudomonas, Xanthomonas, Brevibacterium, and Microbacterium, which are considered to synthesize metabolites with known antimicrobial and antioxidant activity [24][25][26]. Endophytic fungi are considered as a good source of antibiotics and anticancer drugs extracted from Penicillium, Fusarium spp., Pestalotiopsis jesteri, Chloridium spp., Beauveria bassiana, and Metarhizium anisopliae [27][28][29]. Series of bioactive chemical compounds have been isolated from endophytes, investigation has revealed their medicinal activity in several disease models and therefore could be an excellent source of drug for antibacterial, antiviral, antifungal, anticancer, anti-inflammatory, and neuroprotective purposes [30][31][32][33]. With ongoing scientific studies, there is a hope of a finding multipotential role of novel endophytic bioactive molecules against several health impairments, including neurodegenerative disorders [7,[34][35][36]. e present review will discuss the updated and quantified information on bioactive compounds of endophytic fungi and their effects on different ND with promising pharmacological or clinical perspectives.

Methodology
Published literature on the neuroprotective potential of endophytic fungi were collected from different online sources such as PubMed, ScienceDirect, Web of Science, SpringerLink, Wiley online library, and Google Scholar by using specific keywords "Neuroprotective activities of endophytic fungi" and "Bioactive compounds of endophytic fungi and neuroprotection" from 2006 to 2022 (July). Published research and review articles, and book chapters in English were included in this study, whereas duplicate and inappropriate articles related with the topic were excluded from the study.

Endophytic Fungi as a Source of Bioactive Compounds
For centuries, human civilization has greatly depended on plant sources in drug formulations to fight against numerous forms of diseases. Various plant species serve as a major resource for the isolation of diverse active compounds including, alkaloids, phenols, flavonoids, and vitamins, which act on diseases like cancer, diabetes, microbial diseases, neurological disorders, heart diseases, and skin disease [37][38][39][40]. However, in the international market, the demand for active compounds is continuously increasing due to which many plant species are facing severe threats. is problem raises an increased interest among worldwide researchers to find other alternative sources for extraction of the high valued secondary metabolites. In the last few decades, it has been reported that microorganisms integrated with plants, also known as endophytes, can synthesize biologically active compounds which possess promising therapeutic potential [14]. Generally, endophytes are class of microorganisms often actinomycetes, bacteria, and fungi which resides in intercellular or intracellular locations in the plants and show endosymbiotic association with the host plant ( Figure 1) [41,42]. ey play a significant role in synthesis of novel biologically active compounds including phenols, quinones, alkaloids, saponins, tannins, and flavonoids [43]. ese microorganisms are found in almost all plant species, are ubiquitous in nature, and show complex interactions (antagonism, rarely parasitism, and mutualism) with host plants [44]. Endophytes help plants in many ways like enhancing the plant growth and nutrient uptake from the surrounding ( Figure 2). ey are known to colonize different plant parts including leaf segments, fruits, roots, stems, buds, seeds, petioles, inflorescence, and also in deceased and hollow plant cells [45][46][47].
Fungi are an important group of heterotrophic organisms which have complex lifecycle with multiple stages and interestingly they are observed to have a symbiotic relationship with autotrophs. ey are also referred as symptomless symbionts which reside within the plant tissues of angiosperms, gymnosperms, ferns, and mosses [27,48]. According to life history and phylogeny, endophytic fungi are grouped into two: clavicipitaceous and nonclavicipitaceous. Clavicipitaceous endophytic fungi are restricted to cool regions and cause infection in some grasses; however, nonclavicipitaceous are confined to the Ascomycota or Basidiomycota group and are present in vascular and nonvascular plant tissues [49,50]. Endophytic fungi help host plants in nutrient uptake, produce plant growth hormones like auxins, gibberellins, and cytokinins, and aid plants in enhancing their self-defense mechanism [49,51]. e active compounds generated by these fungi are essential for determining the adaptability of both the endophytic fungi and their host plant, especially in harsh environmental conditions, which include biotic and abiotic stresses [52][53][54][55][56][57]. Also, the bioactive compounds by these fungi possess potential applications in the food, cosmetic, agriculture, and medicine industries [58]. Pestalotheol C, an antiviral compound is isolated from Pestalotiopsis theae, an endophytic fungus [59]. Phomopsichalasin, an antibacterial compound which shows significant bactericidal activity against human pathogenic Gram-positive and Gramnegative bacteria, is obtained from Phomopsis sp. and plant host Salix gracilistyla [60,61]. Anticancer and antineoplastic agents such as taxol, vincristine, vinblastine, and camptothecin can be isolated from the endophytic fungus

Neuroprotective Studies and Proposed Molecular Mechanism
Neurodegeneration is defined as a slow and progressive loss of neuronal structure and function in the specified region of the brain that resulted in neuronal cell death [68,69]. By 2040, the ND are estimated to exceed cancer in ranking, as the second major cause of death among the elderly [70]. erefore, it is important to explore therapeutic compounds from natural resources against ND as they possess higher benefits including no/fewer side effects, cost effective, and easily available, over synthesized compounds. Neuroprotective effects of different bioactive compounds isolated from endophytic fungi have been investigated for cure and management of neurodegenerative diseases.
is review highlights the endophytes-derived bioactive compounds and their proposed mechanism of action via different pathways with therapeutic applications.
Recently, bioactive compounds present in endophytic fungi Nigrospora oryzae were screened for their acetylcholinesterase (AChE) and antioxidant activity [71]. Also, one of the isolates from the study, Nigrospora oryzae (GL15) showed maximum AChE as well as antioxidant activity, and the compound (fraction 3) accountable for these activities was identified as quercetin based on analyses using ultraviolet spectrophotometers (UV), fourier-transform infrared spectroscopy (FTIR), electrospray ionisation mass spectrometry (ESI-MS), high-performance liquid chromatography, (HPLC) and proton nuclear magnetic resonance ( 1 H NMR). Additionally, the extract exhibited antidementia-like activity which led to learning and memory shortfalls through the AChE-mediated mechanism in the scopolamine model. e extract also enhanced the scopolamine-induced modulation in the cholinergic pathway and as well as triggered decrease in the activity of AChE and restoration of cytoarchitecture of hippocampus [71]. While in another study by Hou and group [72], a total of seven dibenzopyrone phenolic derivatives including alternariol, alternariol 5-O-methyl ether, altenusin B, altenuene, altenusin, alterlactone, and dehydroaltenusin were extracted and identified using different spectroscopic methods from the endophytic fungi, Alternaria alternate. In this study, the compounds altenuene, altenusin, alterlactone, and dehydroaltenusin demonstrated significant neuroprotective effects against oxidative injuries by acting as potent activators of nuclear factor-erythroid derived 2-like 2 in PC12 cells. ese compounds induced the nuclear accumulation of Nrf2, promoted the expression of Nrf2-governed cytoprotective genes, as well as increased the cellular antioxidant capacity [72]. Al-Qaralleh [73] in their study evaluated the crude extract of Fusarium spp., an endophytic fungi, and isolated OQ-Fus-2-F collected from the stem of Euphorbia plant. e crude extracts were tested for biological activities including antibacterial, antioxidant, and AchE inhibitory activity. e isolate OQ-Fus-2-F showed moderate biological activity in terms of antioxidant activity (ABTS : IC 50 � 37.5 ± 3.5 µg/mL and DPPH : IC 50 � 191.3 ± 17.6 µg/mL) and AChE inhibition activity (IC 50 � 177.0 ± 13.7 µg/mL), respectively [73].
ese compounds showed protective activity against glutamate-induced cytotoxicity in HT22 cells. Among these compounds, Υ-pyrone, javanicin, and fusarubin showed the acceptable neuroprotective activity in a dose-dependent manner. However, fusarubin at 12.5 µM concentration displayed highest cell viability of 90.7 ± 4.5% in HT22 cells, it also possess strong DPPH scavenging activity [76].
A research group from China isolated and identified a total 26 endophytic fungi from the leaves, stems, and roots of the wild Huperzia serrate. Among these fungi, Fusarium verticillioides, Fusarium oxysporum, Mucor racemosus, Mucor fragilis, and Trichoderma harzianum produce  Figure 3: (a) Bioactive compounds previously isolated from endophytic fungi. Figure 3 (b) Bioactive compounds previously isolated from endophytic fungi.

Evidence-Based Complementary and Alternative Medicine
Huperzine A, a potent AChE inhibitor against AD, using thin layer chromatography (TLC), HPLC, and LC-MS/MS analyses [77]. However, in another study, Zaki and coworkers from Egypt also isolated and identified some endophytic fungi from the different parts of wild Huperzia serrata, which were evaluated for their anti-AChE activity and Huperzine A production [78]. However, among all 11 isolates (AGF040 to AGF050), only four endophytic fungal isolates (AGF041, 42, 44, and 46) of Alternaria spp., Penicillium spp., and Colletotrichum spp. genera displayed AChE inhibition activity (more than 50%) however, endophytic fugal isolate Alternaria brassicae AGF041, demonstrated the maximum inhibitory activity (75.5 ± 0.5%), and Huperzine A production, respectively [78]. Glutamate, an essential neurotransmitter of CNS at high concentration can cause ND. Several studies reported that neuronal cell death mediated by glutamate can cause various ND, including AD, brain trauma, cerebral ischemia, PD, epilepsy, and stroke [79][80][81]. High glutamate concentration results in excitotoxicity and high level production of reactive oxygen species (ROS), which further triggers neuronal cell death [82,83]. It is thought that diseases associated with glutamatergic dysfunction produce disruption of calcium homeostasis, increased the production of nitric oxide and increases the oxidative stress resulting in programmed cell death and causing progressive neurodegeneration [79]. Regulating the glutamate levels can lower the excitotoxicity, ROS production and irregular influx of calcium may be an effectual therapeutic strategy for ND [84,85]. Neuroprotective compounds have ability to inhibit glutamateinduced mitochondrial fission by regulating abnormal calcium influx and calcineurin-dependent dephosphorylation of Drp-1 through scavenging mitochondrial and cytosolic ROS [86]. Endophytic bioactive compounds such as Υ-pyrone, fusarester D, karuquinone B, javanicin, solaniol, anhydrooxysproridinone, fischerin, and fusarubin, showed protective activity against glutamate induced cytotoxicity in in vitro models [87]. e proposed molecular mechanism of action of the neuroprotective compounds isolated from endophytes against glutamate induced neuronal cell death is presented in Figure 4.
Bang et al., identified total of nine bioactive compounds, namely, sartorypyrone E, sartorypyrone A, cyclotryprostatin B, fumitremorgin B, fumitremorgin A, aszonalenin, acetylaszonalenin, fischerin and pyripyropene A, by using IR, UV, 1 H NMR, and 1 C NMR techniques from the Neosartorya fidcheri JS0553 endophytic fungi isolated from Glehnia littoralis [87]. e protective effects of these bioactive compounds against HT22 cells were investigated on glutamate induced cytotoxicity. e result showed that among all the compounds, fischerin displayed the most significant neuroprotective effects in HT22 cell death induced with glutamate via inhibition of ROS, Ca 2+ , and phosphorylation of mitogen activated protein kinase (MAPKs) (via JNK, ERK1/2, and p38) [87]. In another study [88], five unique cyclic depsipeptides including colletotrichamide A, colletotrichamide B, colletotrichamide C, colletotrichamide D, and colletotrichamide E, with neuroprotective effects were isolated and identified from the endophytic fungi Colletotrichum gloeosporioides JS419 (inner tissue of Suaeda japonica). ese compounds were tested for their protective effects against glutamate-induced HT22 cell death in which colletotrichamide B, colletotrichamide C, and colletotrichamide E showed protective effects, while colletotrichamide C displayed 100% viability (at 100 µM) [88].

Conclusion and Future Prospects
e global diversity of endophytic fungi is far from being accessed, and these endophytic fungi are considered as a metabolic factory capable of unique bioactive compound production. is type of chemical diversity is important for the screening of novel bioactive compounds targeting different types of diseases, which allows them to act as a prototype compound for the development of new specific drugs. e present manuscript is focused on describing "endophytic fungi as a source of bioactive compounds and their invitro neuroprotective activities." e literature survey clearly demonstrated that endophytic fungi and their bioactive compounds played an important role in neuroprotective studies via different pathways, and showed significant results. Furthermore, the isolated active compounds need to be elucidated and authenticated by in-vivo studies as well as clinical studies. Since most of the reported studies are limited to the in-vitro screening, future clinical trials should be conducted to assess the safety issues of these bioactive compounds in the human body in terms of different biological activities.

Data Availability
All data are included within the text.

Conflicts of Interest
e authors declare that they have no conflicts of interest.