Sulforaphane attenuates postnatal proteasome inhibition and improve spatial learning in adult mice

Abstract
Proteasomes are known to degrade proteins involved in various processes like metabolism, signal transduction, cell-cycle regulation, inflammation, and apoptosis. Evidence showed that protein degradation has a strong influence on developing neurons as well as synaptic plasticity. Here, we have shown that sulforaphane (SFN) could prevent the deleterious effects of postnatal proteasomal inhibition on spatial reference and working memory of adult mice. One day old Balb/c mice received intracerebroventricular injections of MG132 and SFN. Sham received an equal volume of aCSF. We observed that SFN pre-administration could attenuate MG132 mediated decrease in proteasome and calpain activities. In vitro findings revealed that SFN could induce proteasomal activity by enhancing the expression of catalytic subunit-β5. SFN pre-administration prevented the hippocampus based spatial memory impairments during adulthood, mediated by postnatal MG132 exposure. Histological examination showed deleterious effects of MG132 on pyramidal neurons and granule cell neurons in DG and CA3 sub-regions respectively. Furthermore, SFN pre-administration has shown to attenuate the effect of MG132 on proteasome subunit-β5 expression and also induce the Nrf2 nuclear translocation. In addition, SFN pre-administered mice have also shown to induce expression of pCaMKII, pCreb, and mature/pro-Bdnf, molecules which play a crucial role in spatial learning and memory consolidation. Our findings have shown that proteasomes play an important role in hippocampal synaptic plasticity during the early postnatal period and SFN pre-administration could enhance the proteasomal activity as well as improve spatial learning and memory consolidation.

1.Introduction
Synapse formation during initial stages of brain development as well as synaptic plasticity in adulthood is essential for proper nervous system functioning. It has been observed that nervous system build more synapses than it actually required during development [1]. Numerous studies have revealed that proteasome mediated protein degradation play an important role during pruning of extra synapses [2]. It has been shown that most of the connections attain maturity in the hippocampus during 2-3 postnatal weeks [3, 4]. Hippocampal development is very crucial for spatial learning and memory formation in rodents [5, 6]. Injuries to the brain during this period have shown to be detrimental for cognitive functions and impart behavioural changes due to modifications in brain structure [7]. Studies in both rodents, as well as humans, have shown that spatial memory is associated with hippocampus [8]. Several reports have suggested that even slight damage to hippocampus could severely impair spatial memory in rodents. It has been shown that dorsal hippocampal lesions involving approximately one-third of total hippocampal volume had a substantial effect on spatial learning in the water maze [9, 10]. Although numerous studies have focused on effects of hippocampal damage in adulthood, not much has been explored what would happen to the hippocampal functioning if proteasome activity gets impaired during postnatal period.

Several reports have shown that protein synthesis is essential for long-term synaptic plasticity. However, how ubiquitin-proteasome mediated protein degradation involved in synaptic plasticity is not completely understood. Development of cell-permeable proteasome inhibitors greatly enhanced the knowledge of proteasome’s physiological roles in mammalian cells. Although proteasome has three active sites, inhibition of each site is not required to reduce protein degradation function. It has been shown that inhibition of only chymotrypsin- like site (Psmb5 subunit) is sufficient to reduce the rates of protein breakdown [11]. One of the most commonly used inhibitors of proteasome is MG132 (Z-Leu-Leu-Leu-al). It is not only more potent than other inhibitors [12], but also more selective, as it has been shown that inhibition of calpains and cathepsins requires at least 10-fold higher concentrations [13]. Recent studies have shown that ubiquitin-proteasome pathway could regulate short-term synaptic plasticity [14]. Moreover, neurotransmitter receptors along with some postsynaptic density proteins have shown to be degraded by ubiquitin-proteasome machinery [15].

In mice, proteasome mediated protein degradation is required for spatial memory consolidation as well as reconsolidation in the hippocampus [16]. Bilateral injection of lactacystin (proteasome inhibitor) into CA1 region of rat hippocampus has shown to block the long-term memory formation in inhibitory avoidance test. Furthermore, involvement of ubiquitin- proteasome mediated degradation has also been confirmed by increased ubiquitination of synaptic proteins in the hippocampus of trained rats [17]. These reports suggest that a highly regulated protein turnover is essential for consolidation and reconsolidation of memories. In addition, several reports have shown that decreased proteasome activity is related to various neurodegenerative disorders like Parkinson’s and Alzheimer’s disease, which showed abnormal accumulation of polypeptides in the brain and leads to neuronal death [18, 19]. In addition, it has been shown that aggregation of proteins, polysaccharides, and lipids are commonly observed in major early-onset neurodegenerative diseases [20] and also in many neurodegenerative diseases of childhood [21]. These cellular conditions are often referred to as “proteinopathies.” Terminally differentiated cells like neurons have shown to require effective cargo targeting, transportation, and degradation to maintain intracellular homeostasis under basal conditions [22]. Therefore, we wanted to check whether postnatal proteasome inhibition could induce deleterious effects on neurons during later stages of life.

Studies have shown that various naturally occurring dietary phytochemicals could play significant roles in maintaining health of neurons [23]. Sulforaphane (1-isothiocyanato-4- methylsulfinylbutane), a hydrolysed product of glucosinolate glucoraphanin, mainly present in vegetables belong to the Brassica family (e.g., broccoli, cabbage, watercress, Brussels sprouts). Recently it has been shown that sulforaphane (SFN) could induce expression of cytoprotective genes through nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. Until today mechanism of action of SFN is not clearly understood, however various reports have shown that it has anti-inflammatory, anti-oxidant, and anti-cancer activities [24]. Recently, it has been shown that SFN could inhibit neuronal histone deacetylase activity and induce Bdnf expression independent of Nrf2 activation [25]. SFN has shown to induce proteasome activity by overexpressing the catalytic core subunit Psmb5 in murine neuroblastoma cells and therefore may prevent aggregation of damaged proteins associated with various neurodegenerative disorders [26]. Moreover, it has been shown that SFN can enhance NGF-induced neurite outgrowth in PC12 cells through Nrf2 activation, suggesting that SFN is capable of enhancing the neuronal plasticity [27].

In the present study, we have investigated the effects of postnatal proteasomal inhibition during early stages of adulthood. As SFN is cytoprotective and could also induce catalytic activity of proteasome, we wanted to check whether it could attenuate the effects of MG132 induced proteasome inhibition. Our results have shown that SFN could increase proteasome activity by increasing the expression of subunit-β5. We found that SFN administration could induced the Nrf2 activation and prevented the deleterious effects of postnatal proteasome inhibition on hippocampal development and also improve spatial reference as well as spatial working memories during adulthood. Moreover, SFN treatment has also enhanced the expression of CaMKII, Creb, and Bdnf, which play a central role in learning and memory consolidation. Taken together, these results suggest that SFN has the potential to attenuate the effects of postnatal proteasomal inhibition.

2.Materials and Methods
Primary antibodies [Bdnf (P-14): sc-33905; β-Actin (AC-15): sc-69879; Creb (C-21): sc-186; pCreb (Ser 133): sc-101663; CaMKII (H300): sc-13082; p-CaMKIIα (Thr 286): sc-12886;spectrin αII: sc-48382; Nrf2 (H300): sc-13032] were purchased from Santa Cruz Biotech Inc. (Santa Cruz, CA, USA). Psmb5 (D1H6B) Rabbit mAb (12919S) was procured from Cell Signaling Technology Inc. (USA). Secondary antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Proteasome Activity Assay Kit (K245) was procured from BioVision, Inc. (Milpitas, CA, USA). Fura-2, AM (F1201) was procured from ThermoFisher Scientific (MA, USA) Trizol reagent (Life Technologies, Carlsbad, CA, USA), SYBR® Green JumpStart™ Taq ReadyMix™ (S4438), MG-132 (C2211), SFN (S4441) and all other chemicals used in the present study were of analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO, USA). IMR32 cell line was purchased from National Centre for Cell Science (NCCS), Pune.An IMR-32 neuroblastoma cell line was used for in vitro analysis. Neuroblastoma cells are obtained from neural crest and serve as an extensively studied model for neuronal differentiation. IMR-32 cells were plated at a density of 104 cells/well in poly-L-lysine coated 96-well culture plates in DMEM supplemented with 10% FBS, penicillin (50 IU/ml), streptomycin (50 µg/ml), 2 mM glutamine, and 1 mM sodium pyruvate. The cultures were maintained at 37°C in a humidified CO2 incubator. IMR-32 cells were pre-treated with different doses of SFN (1µM, 5µM and 10µM) and subjected to MTT assay.

Results showed that 5µM of SFN provide maximum viability and proteasome activity against 1µM ofMG132. Moreover, higher concentration i.e. 10µM of SFN was found to be toxic. Therefore, a dose of 5μM SFN was selected for subsequent experiments.BALB/c mice were mated to generate littermate for the present study. Mice were housed and bred at the local animal house facility. Postnatal day one (P1) pups were selected for performing experiments. Mice were fed with standard mouse pellet diet (Ashirwad Industries; Kharar, India) and were given water ad libitum. All efforts were made to minimize both animal number and distress within the experiments. All the protocols followed in the present study were approved (PU/IAEC/2014/S32) by the Institutional Animal Ethics Committee of the University.Mice pups were cryo-anaesthetised for two minutes. A sterilized glass micropipette attached to 10 µl Hamilton syringe through a long tube was used to inject SFN, MG132 or artificial cerebrospinal fluid (aCSF) as described by Glascock et al. [28]. The syringe was inserted 2.0 mm deep, 0.25 mm lateral to the sagittal suture and 0.50–0.75 mm rostral to the neonatal coronary suture. Under sterile conditions, 2 µl of aCSF (140 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1.2 mM Na2HPO4, adjusted to pH 7.4.) or SFN (5 µM) and MG132 (1 µM) were injected into P1 pups brain.The proteasome activity was measured as per manufacturer instructions with slight modifications. Briefly, brains were isolated from mice pups after 24 h of MG132 administration. Proteasome activity was measured in samples by addition of fluorogenic AMC tagged peptide substrates in assay buffer followed by 30 min incubation at 37 °C. The reaction was stopped by adding 125 mM sodium borate buffer (pH 9.0) containing 7.5% ethanol.

Fluorescence released by AMC was read at 360 nm (excitation) and 460 nm (emission) wavelengths in the multimode plate reader (Tecan Infinite M200 PRO). Proteasome activity was normalized with protein concentration and expressed as a percentage of activity relative to DMSO control.Intracellular free calcium (Ca2+) levels were estimated according to the method of Luo and Shi [29]. The homogenous brain cell suspension from pups was incubated with Fura-2 AM at37°C with gentle shaking. The Fura-2 loaded suspension was centrifuged for 10 min, the pellet was washed once with Ca2+-free buffer and was centrifuged again. Aliquots of the washed suspension were diluted with buffer and pre-incubated for 6 min at 37°C. The intracellular free Ca2+ concentration was determined by measuring the fluorescence at 340nm/380nm excitation and 510 nm emission. Maximum fluorescence (Fmax) was measured by lysis with 20% SDS or 2% Triton X-100, followed by the addition of 0.5M EGTA (pH 8.0) to obtain minimal fluorescence (Fmin).Ca2+ levels were measured according to the formula (The Grynkiewicz equation):Where, Kd (for Ca2+ binding to Fura-2 at 37°C) = 225 nM, R = 340/380 ratio, Rmax = 340/380 ratio under Ca2+-saturating conditions, Rmin = 340/380 ratio under Ca2+-free conditions, and Sfb = ratio of baseline fluorescence (380 nm) under Ca2+-free and -bound conditions. (The Kd for Ca2+ binding to Fura-2 decreases with decreasing temperature).It is a test of spatial learning that relies on distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform [30]. The apparatus consists of a water tank 100 cm in diameter with a platform submerged one cm below the surface of the water and placed in one of the imaginary quadrants. The three months old mice were acclimatized to the MWM apparatus five min each for three days. This was followed by four days of active training to reach on a hidden platform. On each training trial, mice were placed into the water with their nose facing the wall of tank at a maximum distance from the hidden platform.

The final acquisition was recorded after the last day of training i.e. on fifth day, under exactly same experimental conditions. The escape latency (i.e., time to locate the platform), path length (i.e. distance covered to reach the platform) and path efficiency (i.e. an index of efficiency of path taken by animal to get from first position in the test to last position, a value of one indicate perfect efficiency. It can be calculated by measuring straight line distance between start position and last position divided by total distance travelled by animal during the test) to reach hidden platform were recorded using AnyMaze video tracking software (Stoelting Co. Wooddale, IL, USA).The Y-maze apparatus has three arms made of black plastic joined in the middle to form a ‘‘Y’’ shape. The walls of the arms were seven cm high, enabling the mouse to see spatial cues. The inside of the arms was uniform without any intra-maze cues. This test is validated as a hippocampally relevant spatial task and based on published protocols with slight modifications [31]. Briefly, each mouse of the test group was placed into start arm and allowed to explore the maze with one closed arm for five min (training trial). After one hour, each mouse of the test group was again placed in the start arm. The mice had excess to all three arms and were allowed to explore the maze for five min (test trial). The number of entries into and the time spent in each arm were recorded.Paraffin-embedded brain sections were deparaffinized with absolute xylene and dehydrated in a sequential ethanol dilution.

The sections were then rehydrated with deionized water followed by staining with Hematoxylin dye for 20 min at room temperature. The sections were again rinsed with deionized water and fast dipped in 1% acid-ethanol solution. After rinsing with deionized water, sections were stained with Eosin dye for 30 s at room temperature. The sections were mounted with DPX, covered with a coverslip and observed under light microscope. Similarly, the paraffin embedded brain sections were first deparaffinised, dehydrated and then rehydrated followed by a brief (five min) cresyl violet staining at room temperature. The sections were washed with deionized water, mounted with DPX and observed under EVOS FL Auto Imaging System (Life Technology, USA). Analysis of morphological features on whole cross sections was performed using the H&E colour deconvolution plugin of ImageJ (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/), which separates the Hematoxylin component and the Eosin component. Normal, undamaged cells were represented by the Eosin component and this area can be measured by thresholding.Total RNA was isolated from hippocampi dissected from mice brains using Trizol reagent (Life Technologies, USA) according to manufacturer’s instructions. cDNA was synthesized using MMLV Reverse Transcriptase kit (Life Technologies, USA) and RT-PCR performed with SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma, USA) with the primers listed in Table 1. Primer specificity was assessed by NCBI BLAST analysis and verification from dissociation curve, Tm and amplicon length. The PCR cycling program was: 95°C for fivemin, followed by 35 cycles of 95°C for one min at respective annealing temperatures (Table 1), finally 72°C for 10 s. Target RNA expression levels were normalized to the housekeeping control (Eukaryotic elongation factor 2, eEF2).

Relative changes in mRNA expression levels between control and MG132 samples were calculated as described by Pfaffl [32].Whole-cell protein extracts were prepared, resolved by electrophoresis and transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) as described by Towbin et al. [33]. After blocking with 5% (w/v) non-fat milk in PBS-T (Phosphate-buffered solution pH 7.6/0.005% Tween-20) the membranes were incubated overnight with respective primary antibody (1:1000) diluted in a blocking buffer. The primary antibody was followed by two hour incubation with relevant horseradish peroxidase-conjugated secondary antibodies (1:2000). All subsequent procedures were performed in a dark room. The membrane was incubated with ECL Substrate (containing equal volumes of peroxide reagent and luminol reagent in a 1:1 ratio [Clarity™ Western ECL Substrate, BioRad, Hercules, CA, USA]) for one min. The membrane was then wrapped with a plastic sheet and exposed to X-ray film at room temperature for 30 s to detect the chemiluminescence. The molecular weights of respective proteins were estimated with pre-stained protein markers (Bio-Rad, Hercules, CA, USA). Band intensities were measured by a densitometry analysis of immunoblots with AlphaEaseFC™ software (USA).Paraffin-embedded brain sections were deparaffinized and dehydrated as described earlier. Endogenous peroxidase activity was blocked by incubation with 0.25% (v/v) H2O2 and 0.001% (w/v) sodium azide in PBS for 20 min at room temperature. Sections were boiled in water bath for 15 min in 10 mM Tris-1 mM EDTA buffer (pH 9.0) for antigen retrieval. Slides were blocked with 1% BSA for 15 min and incubated with Psmb5 (1:200) and Bdnf (1:200) at room temperature for four hour.
After being washed with PBS (pH 7.6), the sections were incubated with respective HRP-conjugated secondary antibody (1:400) at room temperature for one hour. Nuclei were counterstained with hematoxylin. The sections were washed with deionized water and mounted with DPX. The sections were visualized under EVOS FL Auto Imaging System (Life Technology, USA).

Image analyses were performed off-line with the help of IHC tool box plugin of ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). Complete information regarding theuse of this plugin has been provided on following web link (https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html).Hippocampal cells (1×106 cells/ml) were fixed in cold and freshly prepared 2% (v/v) paraformaldehyde in phosphate buffered saline (PBS). Paraformaldehyde fixation was performed with gentle agitation during the addition of fixative to prevent cell clumping. Cells were then incubated for 15 min at 4°C, followed by centrifugation at 500g for five min and pellet obtained was then resuspended in PBS. Plasma membrane must be permeated to allow entry of cell-impermeable fluorescent probes. For cell permeabilization, the cells were incubated with 0.1% (v/v) Triton X-100 for five min at 4°C. Finally, samples were centrifuged at 500g for five min and pellet was resuspended in PBS.Hippocampal cells were incubated with anti-Bdnf (1:500) and anti-NeuN (1:500) antibody overnight at 4°C. Next, cells were washed and then incubated with appropriate PE- or FITC- conjugated secondary antibodies (1:1000) for 30 min at room temperature. Unstained cells from each group were used to check autofluorescence. At least 10,000 events were acquired (BD-LSRFortssa, BD Biosciences, San Jose, CA, USA) with user-defined gate. Data were analyzed using FACS Diva 8.0 software.All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using one-way ANOVA followed by Student–Newman–Keuls posthoc test for multiple pairwise comparisons between the groups using SigmaStat 3.5 software. Values with p < 0.05 were considered as statistically significant. Error bars represent SD.

3.Results
One day old (P1) mice pups received an intracerebroventricular (i.c.v.) injection of either artificial cerebrospinal fluid (aCSF) or SFN, six hours before MG132 administration, and sacrificed after 18 h (Fig. 1a). We found that MG132 induce significant (0.65 fold, *p < 0.001) proteasome inhibition in mid-brain of mice pups. However, SFN pre-administration significantly (0.84 fold, #p < 0.001) rescued the MG132 induced proteasome inhibition (Fig. 1b). We observed significant (0.34 fold, *p < 0.001) elevation in the intracellular Ca2+ levels
after MG132 administration, which was prevented by SFN (0.15 fold, #p < 0.001) pre- administration (Fig. 1c). MG132 is also known to inhibit calpain activity, so next, we checked the effect of MG132 on brain αII-spectrin or α-fodrin cleavage property of calpain. Calpain cleaves αII-spectrin (250kDa) into spectrin breakdown products of 150 and 120 kDa. Western blot analyses showed that MG132 administration could reduce calpain activity and therefore αII-spectrin levels were found to be increased (αII-spectrin: 0.77 fold, *p < 0.001) whereas levels of breakdown products were significantly decreased (SBDP150: 0.27 fold, *p < 0.001; SBDP120: 0.29 fold, *p < 0.001). However, SFN pre-administration could significantly induce (0.61 fold, #p < 0.001) αII-spectrin cleavage and thereby its breakdown products were increased (SBDP150: 0.20 fold, #p < 0.001; SBDP120: 0.25 fold, #p < 0.001) (Fig. 1d). These results suggest that MG132 could induce proteasome inhibition in mice pup brains, which could be prevented by SFN pre-administration.

Next, we checked if SFN could prevent the MG132 mediated proteasome inhibition. IMR32 cells were used to understand this mechanism and treated with SFN for six hours before MG132 exposure (Fig. 2a). Morphological assessment revealed that SFN treatment could induce morphological differentiation of IMR32 cells, whereas cells lost their characteristic elongated morphology and become spherical after 18 h of MG132 administration (Fig. 2b). We found that proteasome inhibition could induce significant (0.55 fold, *p < 0.001) reduction in the cell viability. SFN pre-treatment significantly (0.41 fold, #p = 0.002) rescue the cells from MG132 induced death (Fig. 2c). Further, we checked proteasome activity and observed a sharp reduction (0.89 fold, *p < 0.001) in presence of MG132. However, SFN pre- treatment significantly (5.60 fold, #p < 0.001) prevented MG132 mediated proteasome inhibition (Fig. 2d). Next, we assessed the effects of MG132 administration on catalytic core subunits of proteasome either in presence or absence of SFN. Real-Time PCR analyses revealed significant decrease in Psmb5 (0.68 fold, *p < 0.001) and Psmb7 (0.51 fold, *p = 0.005), but no change in Psmb6 (*p = 0.813) mRNA levels when cells were treated with MG132. Psmb5, Psmb6 and Psmb7 mRNAs code for β5, β1 and β2 subunits of 20S proteasome, respectively. SFN pre-treatment not only prevented the MG132 mediated decrease (Psmb5: 6.71 fold, #p < 0.001; Psmb7: 1.76 fold, #p = 0.002) but also enhance the transcription of these catalytic core subunits (Psmb5: 2.59 fold, †p < 0.001; Psmb7: 1.46 fold, †p = 0.001) (Fig. 2e). These results
suggested that SFN could induce transcriptional activation of genes coding for catalytic core subunits.

To check, whether these transcriptional changes were stably translated or not we performed immunofluorescence and western blot analyses of catalytic core β5 subunit. IMR32 cells showed marked reduction in the expression of β5 subunits when treated with MG132. However, SFN pre-treatment could induce the expression not only in presence but also in absence of MG132 (Fig. 2f). Western blot analyses also showed that MG132 treatment could significantly reduce the expression of β5 (0.11 fold, *p < 0.001) subunits whereas, SFN pre- treatment could significantly (β5: 0.36 fold, #p < 0.001) prevent the MG132 mediated decrease (Fig. 2g). These results clearly demonstrated that SFN could induce the translation of catalytic core subunits, and therefore could increase the proteolytic potential of cells. Mice pups (P1) received either aCSF or SFN i.c.v. injection six hours before MG132 administration and assessed modulations in hippocampus-dependent memory functions after three months (Fig. 3a). Morris water maze is commonly used to measure spatial reference memory. Mice were trained to explore a submerged platform in a water tank for four consecutive days and assessed their average speed, escape latency, path efficiency and time spent in exploring each quadrant using video tracking system. The analyses revealed that postnatal MG132 administered mice were unable to locate hidden platform and spent more time in every quadrant before finding the platform when compared to shams (Fig. 3b). Moreover, they had significantly lower path efficiency when compared to shams on last day of training.

Throughout the training sessions, MG132 administered mice showed no significant (TD1, *p = 0.138; TD2, *p = 0.903; TD3, *p = 0.045; TD4, *p = 0.174) change in average speed but showed higher escape latency (TD1, *p = 0.702; TD2, *p = 0.842; TD3, *p = 0.069; TD4: 6.43 fold, *p = 0.002) and substantially lower path efficiencies (TD1, *p = 0.996; TD2: 0.55 fold, *p = 0.004; TD3: 0.59 fold, *p = 0.010; TD4: 0.61 fold, *p = 0.016) than shams (Fig. 3c-3e). These findings suggested that postnatal MG132 administration had significantly affected the spatial reference memory of mice. However, postnatal pre- administration of SFN in mice showed better results in spatial memory test than MG132 administered mice. SFN pre-administered mice showed gradual decrease in escape latency (TD3: 0.45 fold, #p = 0.037; TD4: 0.56 fold, #p = 0.021) with higher path efficiencies (TD3: 0.77 fold, #p = 0.044; TD4: 1.32 fold, #p = 0.021) than MG132 administered mice during training sessions. These results revealed that postnatal pre-administration of SFN could attenuate MG132 mediated spatial memory impairments in adulthood. Next, we performed Y-maze test to analyze the effects of postnatal MG132 administration on spatial working memory (Fig. 3f). Higher tendency to explore novel arm rather than familiar arm suggest mouse has better spatial working memory. We observed significant decrease (0.56 fold, *p = 0.001) in novel arm entries by MG132 administered mice (Fig. 3g). Moreover, they spent significantly more time (0.69 fold, *p < 0.001) and travel more (0.44 fold, *p = 0.047) in familiar arm when compared to shams (Fig. 3h and 3i). SFN pre- administration significantly attenuate the effects of MG132 in adult mice, as these mice travelled more (0.84 fold, #p < 0.05) and had high exploration tendency (0.93 fold, #p < 0.001) in novel arm when compared to MG132 administered mice. Hence, these findings suggest that SFN pre-administration could prevent the MG132 mediated deleterious effects on spatial working memory.

Histopathological examination revealed that postnatal administration of MG132 could impart long lasting effects on hippocampal neurons present in different regions of dentate gyrus (DG) and cornu ammonis (CA). We observed that DG and CA3 hippocampal neurons were severely affected after postnatal proteasome inhibition. DG sub-region showed marked increase in granule cells pyknosis, CA3 neurons present in stratum oriens and stratum radiatum were found to be highly vacuolated whereas, CA3 pyramidal cell layer had shown marked increase in shrunken neurons when compared to sham. CA1 stratum radiatum has shown disarranged neuronal network, however, no visible effect was observed on CA1 pyramidal cells after MG132 administration when compared to sham. H & E staining clearly demonstrated that SFN pre-administration in mice could prevent MG132 mediated detrimental effects and had shown normal neuronal morphology and arrangement in DG, CA3 and CA1 regions (Fig. 4a). These findings indicate that postnatal proteasome inhibition resulted in defective hippocampus development and therefore these mice could not properly receive and process the spatial information whereas, SFN pre-administration could prevent this detrimental effect.

In addition, protein expression of Nrf2 was significantly reduced in nuclear fraction (0.27 fold, *p < 0.001) and increased in cytosolic fraction (0.97 fold, *p < 0.01), in MG132 administered mice. However, SFN pre-administration not only prevented MG132 mediated decrease in Nrf2 levels but also enhanced the nuclear translocation (1.84 fold, #p < 0.001). Moreover, we observed reduced expression in cytosolic fraction when SFN was pre- administered (0.16 fold, #p < 0.01). Next, we checked the expression of β5 subunit in pyramidal neurons of hippocampus. Immunohistochemical analyses showed significant decrease in the expression of β5 (DG: 0.17 fold, *p < 0.001; CA3: 0.27 fold, *p < 0.001; CA1: 0.33 fold, *p < 0.001) subunits in granule cells of DG as well as pyramidal neurons of CA3 and CA1 hippocampus after postnatal MG132 administration. However, SFN pre-administration have shown to prevent the MG132 mediated decrease in the expression of β5 subunits (DG: 0.15 fold, #p < 0.001; CA3: 0.27 fold, #p < 0.001; CA1: 0.51 fold, #p < 0.001), which were nearly comparable to sham (Fig. 4b). These results clearly demonstrate that SFN could enhance proteasome functioning by inducing the synthesis of new proteasomes or enhancing the activity of already existing proteasomes.

Recently, it has been shown that SFN could epigenetically enhance the neuronal expression of Bdnf as well as other important molecules which regulate synaptic plasticity and also prevents neurodegenerative disorder like Alzheimer’s [34]. Real-Time PCR analysis revealed that postnatal MG132 administration could decrease Bdnf mRNA (0.51 fold, *p = 0.011) expression in the hippocampus. However, SFN pre-administration was found to prevent MG132 mediated Bdnf down-regulation (2.42 fold, #p < 0.001) (Fig. 5a). Next, we immunohistochemically assessed the expression of Bdnf in pyramidal neurons of hippocampus. A significant reduction in the Bdnf expression (0.36 fold, *p < 0.001) was observed in CA3 hippocampal pyramidal neurons of MG132 administered mice. SFN pre- administration prevented the MG132 mediated decrease in hippocampal Bdnf expression (1.04 fold, #p < 0.001) (Fig. 5b). Western blot analyses also confirmed immunohistochemical findings and showed decreased mature:pro Bdnf ratio (0.27 fold, *p = 0.003) in the hippocampus of MG132 administered mice. SFN pre-administered mice have not shown signs of imbalance and the mature:pro Bdnf levels (0.38 fold, #p = 0.007) were comparable to shams (Fig. 5c). Further, we wanted to check the levels of Bdnf specifically in hippocampal neurons. Flowcytometric analysis revealed postnatal MG132 administration could induce a significant decrease (0.34 fold, *p < 0.001) in the Bdnf positive hippocampal neurons when compared to shams. However, SFN pre-administration could prevent this decrease in Bdnf positive hippocampal neurons and their number was comparable to shams (0.31 fold, #p < 0.001) (Fig. 5d). CaMKII and Creb activation are known to be regulated by Bdnf, so we checked their expression in the hippocampus. Postnatal MG132 administration resulted in significant decrease in the pT286-CaMKII (0.26 fold, *p < 0.001) and pS133-Creb (0.33 fold, *p < 0.001) levels in MG132 administered mice when compared to sham. However, SFN pre- administration significantly prevented MG132 mediated down-regulation of pT286-CaMKII (0.25 fold, #p < 0.001) and pS133-Creb (0.42 fold, #p < 0.001) expression in the hippocampus of adult mice (Fig. 5e and 5f).

4.Discussion
Proteasome plays an important role during postnatal brain development and any fault in its functioning could induce cognitive impairments in adulthood. Here, we have shown that SFN pre-administration could attenuate the detrimental effects of postnatal proteasome inhibition by enhancing the expression of various catalytic core subunits of proteasome. Moreover, SFN pre-administered mice showed better learning skills in adulthood than MG132 or sham mice. It has been shown that MG-132 could inhibit proteasomal activity within minutes and leads to accumulation of intracellular ubiquitinated proteins [35]. These accumulated proteins could be detrimental during early stages of brain development and therefore proper functioning of this degradation machinery is continuously required. To our knowledge, our data are first to report that SFN could prevent MG132 mediated postnatal proteasome inhibition. Previously, it has been shown that postnatal proteasome inhibition in mice could lead to impairments in hippocampus as well as amygdala-dependent tasks at later stages of life [36]. Apart from proteasome inhibition, MG132 could induce intracellular Ca2+ ion accumulation and also inhibit calpain activity [13, 37]. A previous report has shown a direct relationship between increased intracellular Ca2+ ions and calpain activation in vitro [38]. Interestingly, we found that SFN could promote cleavage of αII-spectrin (250kDa), a substrate of calpains, and hence observed increased levels of SBDP-150 and SBDP-120. These findings have suggested that SFN pre-administration could prevent MG132 mediated proteasome inhibition as well as increased intracellular Ca2+ levels and also helped in maintaining basal calpain activities during early stages of brain development.

In order to understand the mechanism of SFN mediated protective effects, we employed IMR32 cells and subjected to same treatment regimen as received by pups. SFN pre- treatment prevented MG132 induced loss of cell viability as well as proteasome activity. Recently, it has been shown that SFN could promote proliferation and differentiation of neural stem cells [39]. Likewise, we found fine projections coming out of cell soma after SFN treatment. Next, we observed that SFN pre-treatment could not only prevent MG132 mediated decrease but also enhance the transcription of proteasome subunits i.e. Psmb5 and Psmb7. Furthermore, we also observed translational increase in levels of catalytic core β5- subunit when cells were pre-treated with SFN. Proteasome comprises three catalytic subunits in its core namely Psmb5, Psmb6 and Psmb7 having chymotrypsin like, caspase like and trypsin like enzymatic activities, respectively. Out of these three, Psmb5 has tandem AREs (antioxidant response elements) in its promoter region which regulates the transcriptional expression of Psmb5 in response to anti-oxidants. In addition, several putative AREs has been identified in other catalytic subunits and therefore, could provide possible explanation for their SFN mediated transcriptional activation. Recently, SFN has been shown to enhance mammalian proteasome activity through induction of 26S proteasome subunit, PSMB5.

Both induction of PSMB5 and proteasome activation have been linked with antioxidant response of Nrf2 [26]. Therefore, next we checked the expression of Nrf2 in hippocampal region of mice administered with MG132 during postnatal period. Western blot analysis revealed that MG132 administration resulted in decreased nuclear translocation of Nrf2. However, SFN pre-administration not only prevented the MG132 induced decrease but enhanced the nuclear translocation of Nrf2. Our findings are in accordance with previous studies and clearly suggest that SFN could induce the proteasomal catalytic subunit Psmb5 via Nrf2 activation. Next, we assessed whether SFN pre-administration could prevent MG132 mediated spatial learning impairments. Mice which received postnatal MG132 showed difficulty in finding hidden platform throughout the MWM training days. However, SFN pre-administered mice showed better learning skills in MWM test. Moreover, we did not observed alterations in average speed but found a continuous decrease in escape latency and increased path efficiency as the training progressed. SFN pre-administered mice also spent more time in exploring quadrant containing hidden platform. In addition, Y-maze analyses showed that

MG132 administered mice were unwilling to explore new environment. However, SFN pre- administered mice showed attenuation of MG132 mediated impairments in spatial memory as evident from increased number of entries, time spent and distance travelled in novel arm over familiar arm. Our results are in accordance with previous reports which suggested that SFN could attenuate spatial memory impairments after various brain insults [40-42]. As these tests are dependent on proper functioning of hippocampus so, our findings suggest that MG132 must have affected the hippocampal processing during adulthood and SFN could attenuate the effects of postnatal proteasome inhibition.
Further, we assessed whether MG132 induce hippocampal damage after postnatal administration. Hematoxylin and eosin staining showed that granule cells of DG and pyramidal neurons of CA3 and CA1 sub-regions of hippocampus were severely affected in MG132 administered mice. SFN pre-administration prevented the deleterious effects of MG132 as evident from cell morphology and arrangement in DG, CA3, and CA1 hippocampal sub-regions. Numerous models have shown that animals tend to learn the spatial locations very rapidly and DG, as well as CA3 regions, are primarily mediates this processing [43, 44].

Different groups have studied the effects of selective hippocampal damage and shown that CA3 is crucial for retrieval of spatial locations [45]. However, others have suggested that CA3 is not as crucial as CA1 for performance of spatial memory processing. It appears that CA3 may rapidly process spatial information and transfer it to CA1 region for further processing [46, 47]. These findings suggest that impairments in proteasomal degradation during brain development could be detrimental for spatial memory formation as well as its retrieval. Increasing evidence has demonstrated that SFN can protect acute brain damage. SFN administration has shown to reduce infarct volume and increase HO1 expression in rodent stroke model [48]. Moreover, SFN administration post-TBI injury has shown to reduce brain edema in rats [49, 50]. Together, these reports along with our results demonstrate that SFN could be a potential therapeutic agent against postnatal brain injury induced by proteasome inhibition.

Neurotrophins like nerve growth factor, neurotrophins and Bdnf have shown to regulate survival and differentiation of neurons [51]. Bdnf is known to play a crucial role in synaptogenesis, maintain neuronal plasticity and is actively involved in memory formation [51, 52]. We observed a substantial decrease in Bdnf mRNA as well as mature:pro Bdnf levels in hippocampus of MG132 administered mice. However, SFN administration has shown to prevent MG132 mediated decrease in hippocampal Bdnf expression. Flowcytometric analysis also showed more Bdnf positive hippocampal neurons in SFN administered mice. These findings are in agreement with recent findings where SFN has not only increased Bdnf expression but also increased the activation of its downstream effectors like CaMKII and pCreb [34]. Likewise, we also observed significant increase in the expression of pT286-CaMKII as well as pS133Creb in hippocampus. CaMKII activation required Ca2+ and calmodulin, resulting in its autophosphorylation at T286, which further activates various signalling molecules and transcription factors like Creb [53]. CaMKII also mediates neurotrophic-dependent activation of Creb in the dentate gyrus [54]. Further, several reports have indicated that spatial memory formation is strongly associated with hippocampal phosphorylation of Creb [55]. Although which specific function assigned to each hippocampal region is not completely understood, but CA1 region has shown to be associated with dorsal hippocampus in spatial memories consolidation [6, 56]. Our findings indicate that SFN could prevent MG132 mediated effects and promote induction of spatial memory formation by activating various molecules like Bdnf, Creb, and CaMKII.

5.Conclusion
In this study, we reported that SFN administration during postnatal brain development in mice could enhance synaptic plasticity and spatial learning skills by increasing the proteasome activity.