Hericium erinaceus potentially rescues behavioural motor deficits through ERK-CREB-PSD95 neuroprotective mechanisms in rat model of 3-acetylpyridine-induced cerebellar ataxia

Cerebellar ataxia is a neurodegenerative disorder with no definitive treatment. Although several studies have demonstrated the neuroprotective effects of Hericium erinaceus (H.E.), its mechanisms in cerebellar ataxia remain largely unknown. Here, we investigated the neuroprotective effects of H.E. treatment in an animal model of 3-acetylpyridine (3-AP)-induced cerebellar ataxia. Animals administered 3-AP injection exhibited remarkable impairments in motor coordination and balance. There were no significant effects of 25 mg/kg H.E. on the 3-AP treatment group compared to the 3-AP saline group. Interestingly, there was also no significant difference in the 3-AP treatment group compared to the non-3-AP control, indicating a potential rescue of motor deficits. Our results revealed that 25 mg/kg H.E. normalised the neuroplasticity-related gene expression to the level of non-3-AP control. These findings were further supported by increased protein expressions of pERK1/2-pCREB-PSD95 as well as neuroprotective effects on cerebellar Purkinje cells in the 3-AP treatment group compared to the 3-AP saline group. In conclusion, our findings suggest that H.E. potentially rescued behavioural motor deficits through the neuroprotective mechanisms of ERK-CREB-PSD95 in an animal model of 3-AP-induced cerebellar ataxia.

Cerebellar ataxia is a progressive neurodegenerative disorder that is characterised by degeneration of the cerebellum, leading to impaired balance, motor dysfunction, and limb and gait ataxia 1,2 . This disorder involves the altered organisation of cerebellar circuits and connectivity, and is often genetically inherited 3 . Previous studies have demonstrated that dysfunction and degeneration of Purkinje cells in the cerebellum largely contribute to cerebellar ataxia 4,5 . The Purkinje cell dysfunction, which involves the deficiency of Ca 2+ -activated K + channels, has been shown to play a major role in locomotor abnormalities and disruption of motor coordination in an animal model of cerebellar ataxia 6 . The inability of Purkinje cell firing serves as a potential target for improving motor dysfunction in ataxic subjects. Although limited complete treatments are available for some rare forms of ataxia with well-studied biochemical defects 1 , only rehabilitative treatment is available for the majority of cases of ataxia. There is scant evidence to support pharmacological management as an effective treatment to improve cerebellar ataxia 7 . Persistent training with intensive exercises focusing on balance and locomotion can slow down the deterioration of balance and impairment of gait in ataxic patients 8 .
Antioxidants have been thoroughly tested for their efficacy to slow down the progressive deterioration in neurodegenerative disorders. Medicinal mushrooms comprise of an efficient antioxidant machinery due to extraction and nutritional composition of H. erinaceus. A standardised aqueous extract of H. erinaceus (NevGro ® , Batch No. 7H2308X, Ganofarm R&D Sdn Bhd, Tanjung Sepat, Selangor, Malaysia) was used in this experiment. The extract was prepared by boiling fresh fruiting bodies of H. erinaceus in reverse osmosis water for 4 h, filtered, concentrated and spray-dried. It contains 20.66% beta 1,3-1,6 glucan and 0.17% adenosine (Nova Laboratories Private Limited, Sepang, Selangor, Malaysia). Total glucan and α-glucan were determined by the β-glucan assay kit (Megazyme International, Wicklow, Ireland). Adenosine content was analysed and quantified by high-performance liquid chromatography (HPLC) using in house method (Nova Laboratories Private Limited, Sepang, Selangor, Malaysia) 37 .
Experimental design and drug administration. For the behavioural baseline assessments, animals were subjected to accelerated rotarod tests on day − 3 and day − 2. Animals were then injected with either 40 mg/ kg 3-Acetylpyridine (3-AP; Sigma-Aldrich, Missouri, USA) or saline (0.9% NaCl) on day − 2. After 2 days of 3-AP treatment, all animals were again tested using the accelerated rotarod test. On day 1, the 3-AP-injected animals were administered aqueous extract of H. erinaceus intraperitoneally at doses of 10 mg/kg and 25 mg/ kg. The control non-3-AP-treated animals and 3-AP-injected animals were both injected with saline. The behavioural battery included accelerated rotarod test, which assesses motor coordination, conducted on days − 3, − 2, 1, 7, 14, and 21; and the rod test, which measures balance and grip strength, conducted on days 15, and 22 (Fig. 1A).
Behavioural tests. Accelerated Rotarod Test: The test was performed as previously described 38 . Briefly, a rat was placed each of four rods (7 cm lane width) in the Rotarod apparatus (Panlab-Harvard Apparatus, Massachusetts, USA). After four rats were placed into the apparatus, the acceleration program was started, which increased the rotation speed progressively from 4 to 40 revolutions per minute (rpm) (Fig. 1B). The total time spent on the rod (latency to fall) for each rat was recorded. The percentage of deficit was calculated based on the following formula: (X -Baseline) / (X + Baseline) × 100%.
Rod Test: A rod (25 mm diameter) was placed on top of an acrylic box (40 cm high). The rat was placed on the rod along its body length. The time spent balancing on the rod (latency to fall) was recorded for up to 5 min.
Histological assessment. After the behavioural tests, rats were euthanised with sodium pentobarbital (Dorminal, Alfasan International BV, Woerden, Holland) and decapitated. Rats were perfused with 0.9% saline and brains were removed. Half of the brain was immersed in 4% paraformaldehyde fixative solution for 1 day, followed by 15% and 30% sucrose-buffer solution for cryoprotection prior to freezing. The fixed brain was fro- . Rats from each group were placed on the lane of the rotarod apparatus, respectively (B). Rats were trained on the rotarod and baseline performance was assessed on day -3 and day -2 prior to the 3-AP injection (C). Accelerated rotarod evaluation of the motor deficits by latency to fall (Rank) (D), percentage of deficit (Rank) (E), and rod test by latency to fall for the assessment of balance (F). The endpoints were rank transformed, and results were presented as mean individual data points with 95% confidence interval. All  Real-time PCR. The cerebellum and motor cortex regions were dissected for the neurogenesis-and neuroplasticity-related gene expression study. The real-time PCR was performed according to our previously published methodology 42 . Total RNA was isolated from the cerebellum and motor cortex area using TRIZOL (Life Technologies, Carlsbad, USA) and converted into cDNA using a cDNA synthesis kit (Takara Bio Inc., Shiga, Japan www.nature.com/scientificreports/ Statistical analysis. The results were analysed using IBM SPSS Statistics 25. The normality of data distribution for behavioural endpoints was examined using the Shapiro-Wilk test. Several endpoints exhibited significant departure from normality and the behavioural data for each of these endpoints were rank-transformed, followed by nonparametric Kruskal-Wallis test for intergroup comparisons. All significant values for nonparametric tests were adjusted by Bonferroni correction for multiple comparisons. For normally distributed data, the results were analysed using one-way ANOVA (with repeated-measures) and by Bonferroni post-hoc test for multiple planned comparisons, as appropriate. Spearman correlation coefficients with Bonferroni correction were calculated to investigate the relationship between different variables related to cerebellum neuroplasticity genes and/or protein functions, as well as the behavioural measures. Scatter plots were drawn only for specific variables with statistically significant correlations. The methodology of statistical analysis was performed as previously described [53][54][55] . All results were presented in individual data points with 95% confidence interval, and p values < 0.05 were considered significant, as appropriate.

Results
Accelerated rotarod test. In (Fig. 1C). Two days after the 3-AP injection on day 1 (before injection of H. erinaceus), Kruskal-Wallis test showed significant main effects in animals treated with 3-AP compared to the non-3-AP control animals (H 3 = 12.309, p = 0.006; Fig. 1D). Pairwise comparisons with Bonferroni correction revealed significant impairments in the accelerated rotarod test in all three of the 3-AP animal groups (all p < 0.033) compared to the non-3-AP control animals.
In the treatment period of H.E., repeated-measures analysis of accelerated rotarod test showed significant main effects for day (F (3,84)  with Bonferroni correction showed significant decreases of latency to fall in 3-AP animals treated with 10 mg/ kg and 25 mg/kg, or saline compared to non-3-AP control group (all p < 0.04). On day 21, there were no statistically significant differences between 3-AP + 10 mg/kg H.E. and 3-AP + 25 mg/kg H.E. groups compared to the 3-AP + saline group (p = n.s.). Interestingly, our results also showed no differences for 3-AP + 10 mg/kg H.E. and 3-AP + 25 mg/kg H.E. groups compared to the non-3-AP control (p = n.s.), indicating a potential rescue of locomotor abnormalities in 3-AP-induced animals with cerebellar ataxia. However, there was a statistically significant difference in the 3-AP + saline group compared to the non-3-AP control (p = 0.018).

Hericium erinaceus (25 mg/kg) rescued the degeneration of Purkinje cells. A row of uniformly
aligned Purkinje cells was present between the relatively lightly-stained molecular layer and the darkly-stained granular layer in the non-3-AP control group (Fig. 5D). However, some of the Purkinje cells in 3-AP + saline group (Fig. 5C) exhibited a disturbed alignment of Purkinje cells with deformed, irregular, and scattered appearance. Interestingly, 3-AP + 10 mg/kg H.E. (Fig. 5A) and 3-AP + 25 mg/kg H.E. (Fig. 5B) groups showed aligned Purkinje cells with regular and ordinary morphology, which was relatively indistinguishable from the non-3-AP control group. Comparison analysis revealed that the Purkinje cell linear density of 3-AP + saline group (1,348 Purkinje cells per 51,171.87 μm total PC length, 25 sections from 5 animals) was significantly lower   In support of the histological findings, qPCR on transcription factors (calbindin-D28k and PCP4) that are highly specific to the postmitotic neurons of Purkinje cell was performed. One-way ANOVA showed main effects on the gene expression of calbindin-D28k (F (3, 32) = 9.009, p < 0.000) and PCP4 (F (3, 33) = 8.878, p < 0.000) in the cerebellum. Interestingly, we found a significant decrease of gene expression for PCP4 and calbindin-D28k in 3-AP + 25 mg/kg H.E. group compared to the 3-AP + saline and the non-3-AP control groups (all p < 0.001; Fig. 5G,H). Besides, there was also a remarkable decrease of PCP4 gene expression in 3-AP + 10 mg/kg H.E. group compared to the 3-AP + saline group (p = 0.018). The reduction of these transcription factors could possibly be explained by which the RNA was used to synthesise calbindin-D28k and PCP4 proteins for gain-of-function purpose in the 3-AP + 25 mg/kg H.E. treated animals. To further investigate the crucial function of apoptosis played by H. erinaceus treatment in this ataxic model, our result show a significant main effect for caspase-3 (F  Table 2). No correlation was found between the accelerated rotarod behaviour data and the percentage of deficit in 3-AP + saline group (r 2 = 0.896, p = n.s.), suggesting the motor coordination behaviour in the accelerated rotarod was altered by 3-AP injection. Interestingly, we found a significant positive correlation between the gene expression for CREB and TrkB in the 3-AP + 25 mg/kg H.E. group (r 2 = 0.688 p = 0.010), indicating a close link between these two genes in the induced neuroplasticity in the cerebellum after H.E. injection (Fig. 6B). Furthermore, significant correlations were also found between the PSD95 protein expression and the rotarod percentage of deficit by latency to fall (r 2 = 0.344, p < 0.001) in 3-AP + 25 mg/kg H.E. group (Fig. 6C).

Discussion
In the initial experimental design, a dose of 65 mg/kg 3-AP was selected to generate the animal model of cerebellar ataxia based on previous studies 58,59 . Surprisingly, all rats administered 65 mg/kg 3-AP injection died within 24 h. We conducted a pilot study to establish the optimal dose for generating the ataxic animal model using lower doses of 40 and 50 mg/kg 3-AP. Our results showed that animals administered 50 mg/kg 3-AP injection still died within 5-7 days, whereas animals injected with 40 mg/kg 3-AP survived and were accompanied by impairment of behavioural motor coordination and balance (Fig. 1D-F). Therefore, 40 mg/kg 3-AP injection was used in the present study to test the hypothesis of the neuroprotective effects of H. erinaceus in rescuing the behavioural motor deficits in 3-AP-induced cerebellar ataxia.
In this study, animals were trained on an accelerated rotarod on day − 3 and day − 2, and their baseline levels were assessed to ensure that all groups possessed comparable motor function behaviour before induction of cerebellar ataxia. Two days after the 3-AP injection on day 1, animals were subjected to the accelerated rotarod test to examine the effectiveness of 3-AP to induce ataxia. Our statistical analysis using Shapiro-Wilk test demonstrated the behavioural data for accelerated rotarod (days 1, 7, 14, and 21) and rotarod (days 15 and 22) tests were not normally distributed; and therefore, the behavioural results were rank transformed and analysed by nonparametric tests according to our previously reported statistical methodology 53 . Our results demonstrated the neurotoxin 3-AP induced remarkable motor impairments in the accelerated rotarod and rod tests compared to the non-3-AP control animals. After H. erinaceus treatments, we found no significant improvements on the behavioural motor deficits in the 3-AP + 10 mg/kg and 25 mg/kg H.E. groups compared to the 3-AP + saline group. However, we also observed no remarkable differences in the motor impairment of the 3-AP + 25 mg/kg H.E. group compared to the non-3-AP control group on day 21, indicating a potential recovery of motor deficits by higher dose H. erinaceus treatment in this ataxic model. There are limitations in the present study in that no significant effects were observed for H.E. on rescuing the behavioural motor impairments compared to the 3-AP + saline group. Some possible explanations could be the short duration of treatment, as the H.E. injections were administered over 3 weeks, or a higher dosage (e.g., 50 mg/kg) may be required to rescue the behavioural motor deficits. In this study, we have ruled out the possibility of a higher dosage requirement to rescue the behavioural motor deficits. Our unpublished data showed that animals injected with 50 mg/kg H.E. became very sick with significant weight reduction after the second week of injection. Therefore, these animals were humanely euthanised based on the regulations of CULATR and the Association for Assessment and Accreditation of Laboratory Animal Care. The unfavourable outcome in the animals administered 50 mg/kg H.E. was possibly due to toxicity or overdose. This leaves the possibility that prolonged treatment (> 3 weeks) might be needed to observe the effectiveness of H. erinaceus in rescuing the motor impairments. However, the animal model of 3-AP-induced cerebellar ataxia used in the present study has been shown to have temporal behavioural motor deficits, in which the ataxic animals recovered from the neurotoxic effects of 3-AP over an extended experimental period 60  www.nature.com/scientificreports/ has been reported that adult rats with 3-AP-induced motor disturbances recovered from the ataxia with less debilitating abnormal movements at 28-43 days after the 3-AP injection 60 . Given that this 3-AP-induced ataxic model has been widely used in many studies 58,59 , the limitation of temporal motor impairments should be taken into consideration when assessing motor function in this model. In view of this, an alternative approach using a genetic model of cerebellar ataxia may be appropriate to assess the neuroprotective function of H. erinaceus.
To further investigate the underlying neuroprotective mechanism of H. erinaceus, gene expression and protein studies of the effects on neuroplasticity were performed. We found significant increases in neuroplasticity and neurogenesis-related gene expressions of Dcx, Nes, TrkB, and CREB in both the 3-AP + saline and 3-AP + 10 mg/ kg H.E groups. The upregulation of gene expression in these groups could be triggered by the activation of molecular recovery mechanisms upon neuronal death induced by the neurotoxin 3-AP, suggesting a compensatory neuronal regeneration or sprouting of injured axons. This observation was in line with a previous study that showed there were regulatory mechanisms in neural stem cells in injured brain as indicated by increased expressions of Dcx and Nes 61 . Adult neural stem cells can be regulated by various stimuli, and can undergo proliferation as well as differentiation through neurogenesis to replace the damaged or lost neurons 62 . Although stroke-induced neurogenesis has been reported 63,64 , neural stem cells have limited capacity for neurogenesis, which tends to decrease with age as observed in the brains of older patients with stroke 65 . Hence, full recovery from brain injury may not be achievable through neurogenesis. Neurogenesis mechanisms may be activated through upregulating the neurogenesis-related genes after the 3-AP injection in order to compensate the cells lost resulted by the 3-AP neurotoxin. However, the compensatory mechanism might not be sufficiently effective in regenerating mature neurons and recovering the cerebellum, as well as recovering the motor behavioural deficits. In contrast, ataxic rats that received 25 mg/kg H.E. treatment for 3 weeks did not show increases in neuroplasticity and neurogenesis-related gene expressions, with the expressions similar to that of the non-3-AP control. This phenomenon could be resulted from the stronger neuroprotective effects of a higher dose of H. erinaceus, which protected or rescued the cells in the cerebellum from being damaged by 3-AP, and thus the upregulation of the neurogenesis-related genes could be inessential. Our findings suggest that the higher dose of H. erinaceus could possibly rescue motor deficits in 3-AP-induced cerebellar ataxia. In this study, we found no significant differences in the gene expressions in the motor cortex, implying that injection of 3-AP was specifically affecting the cerebellar cortex, particularly the Purkinje cells and other cerebellar morphology [66][67][68] .
To support the findings of gene expression study, Western blot analysis was carried out to investigate protein changes related to neuroplasticity function. We found remarkable increases in pERK1/2, pCREB, and PSD95 protein expression levels in 3-AP + 25 mg/kg H.E. group compared to both the 3-AP + saline group and non-3-AP control group. The increased protein expression levels in 3-AP + 25 mg/kg H.E. group was possibly due to compensatory regenerative mechanisms related to the neuronal loss induced by the neurotoxic effects of 3-AP. In this study, we found no significant changes in neuroplasticity-related proteins between the 3-AP + saline group and non-3-AP control group, indicating the expression levels of ERK-CREB-PSD95 in the non-3-AP control were normal in healthy animals. The results suggest the neuroprotective effects of H. erinaceus could be mediated by the protein expression of pERK1/2, pCREB and PSD95, leading to the enhancement of fundamental neuroplasticity processes including neuronal survival and proliferation, and ultimately rescuing the behavioural motor deficits.
In the histological study, we observed the Purkinje cells had regular morphology with normal alignment in the cell layer of the cerebellar cortex in animals treated with H. erinaceus (10 mg/kg and 25 mg/kg) and non-3-AP control group. However, ataxic rats without H. erinaceus treatment had Purkinje cells with abnormal morphological features of distorted cell bodies and loss of cell continuity (random spatial arrangement at wider distances) in the cerebellar cortex, indicating 3-AP exerted its toxic effects by disrupting the Purkinje cell and/ or the spatial distribution of Purkinje cells in the cerebellum. This observation was in line with the findings by Mohammadi and colleagues, in which animals treated with 3-AP injection exhibited Purkinje cells with random arrangement at larger distances compared to the non-3AP control group 68 . Purkinje cells are the sole output neurons in the cerebellum that receive and integrate inputs from parallel fibres and climbing fibres for motor coordination and balance. Neurotoxic 3-AP causes distinctive lesions in the central nervous system by destroying inferior olivary nuclei and their climbing fibres that synapse with Purkinje cells, which induces cerebellar To further support the histological findings of neuroprotective effects of H. erinaceus on Purkinje cells in the cerebellum, our data showed a reduction of transcription factors for calbindin-D28k and PCP4 genes in 3-AP + 10 mg/kg H.E. and 3-AP + 25 mg/kg H.E. animals compared to the 3-AP + saline group (Fig. 5G,H). The changes in transcript and protein levels of calbindin-D28k and PCP4 are important regulatory mechanisms for post-transcriptional processes of postmitotic neurons formation of Purkinje cells. Although no protein data was provided in the present study to support the notion of decrease in these transcription factors that used to produce the calbindin-D28k and PCP4 proteins, our results clearly suggest that the gain-of-function in H. erinaceus treated animals was mediated through ERK-CREB-PSD95 mechanisms to protect the Purkinje cells from degeneration induced by 3-AP. PCP4 is highly specific for the Purkinje cell and it is a key modulator for calcium signalling in the developing brain of postmitotic neuroectoderm cells 69,70 . Of particular interest, overexpression of PCP4 has been shown to affect the functional development of Purkinje cells with motor skill and learning impairment in the mouse models of Down syndrome during postnatal development 70,71 . Recently, it has been shown that the inhibition of calcium-mediated calpain activation is associated with the pro-survival effects of calbindin-D28K 72,73 . Although understanding of the interaction between calbindin-D28K and the pro-apoptotic protein caspase-3 remains obscure, our study has demonstrated a reduction of caspase-3, a crucial mediator of programmed cell death, in 3-AP + 25 mg/kg H.E. animals compared to the 3-AP + saline group. The anti-apoptotic effects of H. erinaceus could possibly be explained by the inhibition of capase-3 cleavage or calcium-mediated death signalling pathway, which protects against cell death from 3-AP.
To further investigate the effects of H. erinaceus, we conducted a correlation study on the relationships of the behavioural motor coordination and of the gene and protein functions. We found a significant positive correlation between the latency to fall and the percentage deficit by latency to fall in the 3-AP + 10 mg/kg and 25 mg/kg H.E. groups and the non-3-AP control group, but no correlation was found for the 3-AP + saline group, indicating H. erinaceus potentially rescued the behavioural motor deficits in 3-AP treated animals. Furthermore, we also observed a positive correlation in gene expression between the CREB and TrkB, as well as the correlation between PSD95 protein expression level and the percentage of latency to fall in the 3-AP + 25 mg/kg H.E. group, suggesting BDNF/TrkB/CREB and PSD95 have important roles in H. erinaceus rescue of the behavioural motor impairments in 3-AP-induced cerebellar ataxia.
Natural products have been used as traditional treatment without any documented adverse effects and many are still in use today. Culinary and medicinal mushrooms including H. erinaceus have been shown to have a wide range of bioactivities, including extending lifespan and delaying onset of age-related diseases 74 . To our knowledge, there are no reports of natural products that possess neuroprotective effects on acquired cerebellar ataxia, and this prompted us to examine the neuroprotective effects of H. erinaceus and to investigate its mechanisms in a neurodegenerative disorder model. Substantial evidence indicates that intrinsic free radical scavenging contributes to the neuroprotective effects of H. erinaceus 37 . Phenolic acids of H. erinaceus that possess hydroxyl groups have been shown to exert antioxidant properties by counteracting oxidative stress and other detrimental changes in brain tissues. Our findings that H. erinaceus has enhanced neuroplasticity functions were in agreement with previous studies on the neuritogenic and nerve regeneration effects of H. erinaceus 16,20,37,[74][75][76][77] .
Hericium erinaceus has been reported to possess bioactive compounds (e.g. erinacines A-G) that can pass through the blood brain barrier and stimulate NGF synthesis [28][29][30][31] . Of particular interest, the standardised aqueous extract of H. erinaceus used in the present study contains 20.66% beta-glucan and 0.17% adenosine 37 . Betaglucan derived from Lentinus edodes or Shiitake mushroom known as lentinan has been demonstrated to induce long-term potentiation in the rat dentate gyrus, indicating the potential effect of beta-glucan on neuroplasticity Figure 6. Scatter plots displaying the correlations between the variables related to the motor behavioural tests, as well as the cerebellar neuroplasticity-and neurogenesis-related relative gene and protein expressions. Notably, there were significant correlations between the accelerated rotarod behavioural data and the percentage of deficits in 3-AP + 10 mg/kg H.E., 3-AP + 25 mg/kg H.E., and non-3-AP control groups. No correlation was found between the accelerated rotarod behavioural data and the percentage of deficits in 3-AP + saline group (r 2 = 0.896, p = n.s.), suggesting the behavioural motor coordination of accelerated rotarod was altered by 3-AP injection (A). In the 3-AP + 25 mg/kg H.E. group, the gene expression of TrkB was positively correlated with the CREB expression (B), and the PSD95 protein expression was positively correlated with the percentage of deficits by latency to fall (C). The hypothetical mechanism of neuronal cell death induced by 3-AP (left) and pathways of H. erinaceus on the role of pERK1/2-pCREB-PSD95 in neuroplasticity-related mechanisms (right). 3-AP reduces the intracellular concentration of NAD + and interferes with Zn 2+ homeostasis, which increases the Zn 2+ neurotoxicity and lead to neuronal death. Upregulation of neuroplasticity and neurogenesis-related genes including TrkB, CREB, Nestin, and Dcx was stimulated by 3-AP to reverse neuronal death (left). After the injection of 3-AP followed by H. erinaceus, the tyrosine kinase receptors are hypothetically activated by NGF and followed by the recruitment of the cytoplasmic protein, Son of Sevenless (SOS). This triggers the activation of the Ras/guanosine triphosphate complex 86 and initiates cytoplasmic kinase signal transduction cascades 87 . MEK1/2 catalyses the phosphorylation of ERK1/2 at Tyr204/187 mediated by importin-7, followed by translocation from the cytoplasm to the nucleus 88,89 . ERK1/2 acts as an upstream regulator by catalysing the phosphorylation of various cytoplasmic and nuclear substrates that encode transcription factors and gene regulatory proteins, including CREB 87,90 . Additionally, the Ca 2+ influx is through the NMDA receptors, which activates the neuronal nitric oxide synthase, that is mediated by PSD-95 80,81 . The induced nitric oxide activates ERK for the expression of neuroplasticity-related proteins, facilitated by cGMP and PKG (right) 82 78 . It has been shown that (1-3)-beta-glucan activates the influx of Ca 2+ in the NR8383 macrophages through receptor-operated Ca 2+ channels 79 . The influx of Ca 2+ into the cell is through the NMDA receptors, and it eventually stimulates the neuronal nitric oxide synthase with the facilitation of PSD95, when other channels are less effective 80,81 . The induced nitric oxide activates the cGMP-protein kinase G (PKG) and ERK signalling, www.nature.com/scientificreports/ and ultimately, it leads to the increased expression of neuroplasticity-related proteins 82 . It has been shown that adenosine enhanced the production of nitric oxide 79 , and it reduced the neuronal damage by alteration of the lactate dehydrogenase release or deoxyglucose transport in primary cortical or hippocampal cell cultures that subjected to hypoxia or glucose deprivation 79,83 . Moreover, the adenosine receptors (A 1 and A 2A receptors) were found to facilitate neuroprotective function by improving the excitotoxic neuronal damage in cell culture models of ischemia/hypoxia 83,84 . Interestingly, a similar neuroprotective effect of adenosine was also shown to attenuate neuronal cell death after the administration of potassium cyanide, an inhibitor of the mitochondrial respiratory chain, in another model of histotoxic anoxia 85 .
3-AP has been found to reduce the intracellular concentration of nicotinamide adenine dinucleotide (NAD + ) and to interfere with zinc ion (Zn 2+ ) homeostasis. This can be followed by impaired glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, accumulation of dihydroxyacetone phosphate (DHAP), reduction in ATP production and increased expression of neuroplasticity and neurogenesis-related genes to reverse neuronal death. In this study, we found pERK1/2 protein expression was increased after treatment with 25 mg/kg H. erinaceus, and its neuroprotective effects against 3-AP-Iinduced neuronal cell death were mediated by Ras/Raf/ MEK/ERK1/2 signalling pathway and by the regulation of pCREB (Fig. 6D). This observation was possibly due to the active compound of erinacines A-G from the H. erinaceus that enhanced the synthesis of NGF. Hypothetically, activation of tyrosine kinase receptors by NGF results in the recruitment of the cytoplasmic protein, Son of Sevenless (SOS), through intracellular Shc and Grb2 domains, which then activates the Ras/guanosine triphosphate complex 86 and initiates cytoplasmic kinase signal transduction cascades 87 , and MEK1/2 catalyses the phosphorylation of ERK1/2 at Tyr204/187 mediated by importin-7, followed by translocation from the cytoplasm to the nucleus 88,89 . ERK1/2 acts as an upstream regulator by catalysing the phosphorylation of various cytoplasmic and nuclear substrates that encode transcription factors and gene regulatory proteins, including activators of transcription, c-Jun and c-Fos, as well as signal transducers, Elk1, CREB and c-Myc 87,90 . These transcription factors and gene regulatory proteins regulate the expression of proteins involved in survival, proliferation, and differentiation 87,88 . Thus, activation of Ras/Raf/MEK/ERK1/2 cascade protects and rescues Purkinje cells from 3-AP-induced neuronal death (Fig. 6D).
In conclusion, our present findings showed that 21-day intraperitoneal administration of H. erinaceus at the dosage of 25 mg/kg, but not 10 mg/kg, partially rescued behavioural motor deficit in rat model of cerebellar ataxia induced by 3-AP. Our results suggest that higher dose of H. erinaceus potentially rescues behavioural motor deficits through ERK-CREB-PSD95 neuroprotective mechanisms and prevent cerebellar Purkinje cell degeneration in rat model of 3-AP-induced cerebellar ataxia. Further in-depth investigations need to focus on the electrophysiological effects of H. erinaceus on spontaneous neuronal firing and intracellular calcium concentration in Purkinje cells of animal models of cerebellar ataxia.