Prenatal exposure of azadiradione leads to developmental disabilities

doi:10.21203/rs.3.rs-4422540/v1

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Prenatal exposure of azadiradione leads to developmental disabilities

https://doi.org/10.21203/rs.3.rs-4422540/v1

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published 23 Sep, 2024

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Azadiradione is a brain permeable phytochemical present in the seed of an Indian medicinal plant, Azadirachta Indica, well-known as Neem. Recently, this small bioactive molecule has been revealed to induce the expression of Ube3a, an ubiquitin ligase whose loss and gain of function is associated with two diverse neurodevelopmental disorders. Here we report that in uteroexposure of azadiradione in mice result in severe developmental disabilities. Treatment of well tolerated dose of azadiradione into the pregnant dam (at an embryonic day 12 and 14) causes substantial decrease in the body weight of the new-born pups at their early developmental periods along with significant cognitive, motor and communication deficits and increased anxiety-like behaviours. As the animals grow from adolescent to adult, their body weight and many behavioural deficits are gradually restored to normalcy, although, the cognitive deficit persists significantly. Biochemical analysis reveals that the azadiradione prenatally exposed mice brain exhibits about 2-3 fold increase in the level of Ube3a at post natal day 25 along with significant increase some of its target proteins linked to synaptic function and plasticity indicating enduring effect of the drug on Ube3a expression. The prenatally azadiradione exposed mice also display increased number of dendritic spines in the hippocampal and cortical pyramidal neurons. These results suggest that Ube3a might be one of the key players in azadiradione-induced developmental disabilities.

Azadiradione belongs to limonoid class of phytochemicals and one of the active constituents in the seed of Indian medicinal plant known as Azadirachta Indica or Neem. Several parts of this plant are being traditionally used in Indian and African subcontinents to treat numerous acute and chronic infectious and metabolic diseases [1–5]. About 35 structurally diverse limonoids including azadiradione were identified from the Neem seeds, although, the biological effects of these molecules are either not known or very poorly understood [6, 7]. Azadiradione was shown to exhibits anti-inflammatory, anti-nociceptive and neuroprotective properties in various cellular and animal models, even though, the underlying molecular mechanisms are unclear [1, 8–10]. Recently, azadiradione was demonstrated to restore impaired protein homeostasis and delay the Huntington’s disease pathogenesis (HD) in its fly and mouse models [11, 9]. In depth study further revealed that this small phytochemical can not only activate heat shock factor 1 (HSF1), a principal controller of molecular chaperones but also promote the expression of Ube3a, a E3 ubiquitin ligase involved in the clearance of many misfolded proteins including mutant huntingtin that leads to HD[11]. Azadiradione also found to alter the expression of few synaptic activity and plasticity regulating protein like brain derived neurotropic factor (BDNF), parvalbumin and activity regulated cytoskeletal associated protein (Arc) and these effects are appears to be mediated via Ube3a[12].

Ube3a was initially characterized as a E3 ubiquitin ligase of the ubiquitin-proteasome system that is typically involved in selective recognition of substrates for their ubiquitination and subsequent proteasomal degradation[13]. Later, Ube3a also demonstrated to acts as a transcriptional coactivator of steroid hormone receptors[14]. One of the fascinating aspects of Ube3a is its imprinted expression in the neuron and astroglia with preferential maternal specific expression [15, 16]. Loss of activity of the maternally inherited UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorders with characteristics developmental disabilities, autistic features, seizures, lack of speech and inappropriate laughter[17, 18]. Surprisingly, increased expression (duplication or triplication) or over activation (through gain of function mutations) of UBE3A is connected with autism, which is clinically characterised by social impairment and repetitive behaviour [19–21]. Clinical manifestations associated with both loss and gain of function in UBE3A indicates that it plays a crucial role in maintaining optimal synaptic connections in different brain regions and that its level must be precisely controlled throughout the brain development[17]. Any perturbation in Ube3a function during brain development might lead to developmental debilities. In fact, there are series of report implying involvement of Ube3a in regulating synaptic activity and plasticity [22–26]. The Ube3a-maternal deficient mice (AS mice) display various behavioural deficits resembles to AS patients [27, 28]. On the contrary, Ube3a overexpressed mice (3-fold increase) exhibit decreased social interaction and stereotype behaviours that are hallmark features of autism [21, 29]. The number of dendritic spines was decreased in various brain areas of AS model mice, while increased in Ube3a overexpressed/over activated mice[30, 23, 31].

Because azadiradione increased Ube3a expression in the brain, we thought its prenatal exposure might have severe developmental consequences. Indeed, we observed that the treatment of azadiradione (10 mg/kg) into the pregnant dam consecutively at embryonic day 12 and 14 (E12 and E14) results in significant delay in various developmental milestones of the new born pups. The in utero drug exposed young animal shows considerable impairment in their learning and memory function, motor coordination, vocalization and open field behaviour and many of these abnormalities improve as animals grown up from young to adult stage. Biochemical analysis reveals the increased expression of Ube3a along with two other synaptic activity and plasticity regulating proteins (BDNF and Arc) even at 25 days after birth. The drug treated mice also display increased number dendritic spines in their hippocampal and cortical neurons compared to control groups.

Materials

Enhanced chemiluminescence (ECL) kit, BCA protein estimation kit, Trizol reagents and mouse monoclonal anti-β-actin (A5316) were purchased from Sigma. Mouse monoclonal anti-Ube3a (SC16689), anti-Arc (SC-17839) and anti-GAPDH (SC-32233) were procured from Santa Cruz Biotechnology, while rabbit polyclonal anti-BDNF (NBP1-59304) was obtained from Novus Biologicals. RT-PCR and PCR kits were acquired from TaKaRa Biomedicals. Secondary antibodies tagged with Horseradish peroxidase (HRP) were bought from Vector laboratories, while fluorophore-conjugated secondary antibodies were procured from Thermo Scientific. Purification of Azadiradione was described elsewhere [9].

Animals and treatments

Wild type mice (C57 back strain) were maintained in regular animal cages with food and water in optimal quantities. All animal experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC) of Indian Institute of Technology, Kharagpur (protocol numbers: IE-3/NJ-BS/1.21) and guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) were followed. Azadiradione was first dissolved in dimethyl sulphoxide (DMSO) and then diluted with normal saline containing 5% DMSO and then injected intra-peritoneally (10 mg/kg body weight) into timed pregnant dams at E12 and 14. Control timed pregnant mice were treated (at E12 and E14) with same volume of saline containing 5% DMSO.

Behavioural experiments

Novel object recognition test-This test was performed within an open field box measuring 45 × 45 × 20 cm. The test was intended to assess visual recognition memory in mice following a period of habituation and exposure to novel objects. Habituation sessions were managed twice daily for five minutes each over two successive days, allowing the mice to adjust to the empty open field environment. Consequently, in the training phase, two matching novel objects were presented into the open field box, positioned 12 cm apart. Each mouse was given a five-minute exploration period before being returned to the home cage. The duration of interaction with each object was recorded through a wall mounted camera. After a 24-hour interval, mice were reintroduced into the same open field box and this time comprising two familiar objects. After a five-minute exploration period, one of the acquainted objects was swapped with a new one, and mice were allowed another five minutes to freely investigate both objects. The amounts of time spent with both acquainted and new objects were recorded. The preference for discovering the novel object was determined by dividing the time spent in discovering the novel object (T_novel) by the total exploration time of both familiar and novel objects (T_novel + T_familiar) and expressed as the recognition index (T_novel / (T_novel + T_familiar)).

Radial maze test-Learning and memory assessment was performed using eight-arm radial maze apparatus. Mice, particularly from age groups of P25 and P55 were selected for the study and habituated to the behavioural study room seven days prior to the test session. The eight-arm radial maze was set up, consisting of a central arm and eight arms extending outward. During the training period, mice underwent 5 days of training with four arms baited for reference memory training and all eight arms baited for working memory training. Each drill session comprised of two trials lasting for 10 minutes each, during which mice were allowed to explore all arms baited with food separately for working as well as reference memory test. Subsequently, a test session was conducted (consecutively for 5 days) to assess both working and reference memory, wherein mice were placed at the central arena of the maze, and their revisits to baited and non-baited arms were recorded. Errors in memory were quantified by measuring the number of times mice returned to previously entered arms during the same trial for working memory and the number of times mice come back to non-baited arms for reference memory.

Open-field test-This test was carried out to evaluate the locomotion and anxiety-related behaviour of mice. Each mouse was positioned in the central arena of an open-field box (45 × 45 × 20 cm) and permitted to discover spontaneously for a period of 5 minutes. The open-field arena was visually partitioned into central and outer zones for analysis purposes. Movement patterns of the mice were captured using an overhead camera, enabling the monitoring of their exploratory behaviour. All the data was analysed using the All-maze video-tracking software, facilitating comprehensive evaluation of mouse behaviour in the open field environment.

Clasping behaviour -In the clasping test, mice were suspended by their tails above 10 cm distance from the cage floor for a maximum duration of 30 seconds, and the time to bring their limbs firmly together was noted. Clasping scores were assigned based on specific time intervals 0–5 sec- 4, 5–10 sec-3, 10–15 sec-2, 15–30 sec-1.

Gait analysis-Footprint gait examination was performed by dipping the hind paws of the mice in a non-toxic colourful ink. Mice were taught to familiarise by walking inside a glass shaft lined with white paper. Subsequently, footprints left by the mice on the white paper lining of the tunnel were analysed to measure parameters such as stride length and width.

Vocalization study-Ultrasonic vocalizations (USVs) are spontaneously emitted by pups when isolated from their mother or separated from littermates and introduced to a new environment within first two weeks of life[32, 33]. Pup isolation calls (PICs) typically fall within the frequency range of 20 kHz to 120 kHz. In this experiment, individual pups were removed from their mother's cage and placed into separate open-surface glass containers (measuring 10 cm × 8 cm × 7 cm) containing fresh bedding. PICs were recorded (using 1/4" microphone paired with amplifier) from the isolated pup, digitized at a sampling rate of 375 kHz on P5 to P11. Each pup was returned to their mother’s cage after 5 minutes of recording. The glass container was sanitized using 70% ethanol after each recording to eliminate any olfactory cues. Additionally, the bedding was changed for the next pup in the experiment sequence. All PIC recordings, collected using the same procedure for both vehicle and azadiradione prenatally exposed pups, were further analysed using MATLAB. Syllables were extracted from the wav file using VocalMat software. VocalMat classifies the syllables into eleven categories. However, we used a broader classification scheme as used by us earlier () with 5 categories that are based on pitch jumps and harmonicity. Down FM, Up FM, Flat, Chevron, Reverse Chevron, Complex are considered as ‘Single’ category (S-type) syllable. Noise and Short syllables were considered as ‘Noise’ category (N-type). Step Up and Step Down were considered as Jump syllables (J-type). Harmonic syllables were unchanged and syllables that were Two Steps or Multiple Steps were considered as ‘Other’ syllables (O-type). Further due to high number of noise detected as syllables by VocalMat, syllables that were very short duration (< 30 ms) were excluded and syllables detected that were greater than 350 ms were rejected due to the absence of the possibility of syllables of such duration and represent a general sustained increase in background noise.

Quantitative real-time RT-PCR

Total RNA was extracted from the azadiradione treated brain samples (cortical and hippocampal regions) using Trizol reagent. The cDNA was made from each sample and then subjected to quantitative real-time PCR (using ViiA7 real time PCR system, Applied Biosystems) for Ube3a, Arc and BDNF with power SYBR Green PCR master mix. RT-PCR products were standardized with 18S RNA as an internal control. Primer sequences for Ube3a, BDNF, Arc and 18S RNA are as follows: Ube3aF, 5'-CATACCTGAGTCCAGCGAATTA-3'; Ube3aR, 5’-ACGCCAAGTTCGGTTTCT-3'; BDNFF, 5’-GGCTGACACTTTTGAGCACGTC-3’; BDNFR, 5’-CTCCAAAGGCACTTGACTGCTG-3’; ArcF, 5’-ACCTGACATCCTGGCACCTC-3’, ArcR, 5’-GTGGTGATGCCCTTTCCAGA-3’; 18SF, 5’-GAGGGAGCCTGAGAAACGG-3’; 18SR, 5’-GTCGGGAGTGGGTAATTTGC-3’. Data were analysed and expressed as fold change.

Immunoblotting experiments

After sacrificing the mice through cervical dislocation, cortical and hippocampal areas of the brain were carefully separated out, quickly frozen into liquid nitrogen and stored at -80°C. Equal amounts of each sample were then homogenised in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and complete protease inhibitor cocktail) and were retained on ice for 30 minutes. Homogenized samples were centrifuged at 15,000 x g for 10 minutes, supernatants were obtained, and amount of protein in each sample was assessed by BCA technique and kept at -80°C in multiple tubes. Different protein samples were mixed with SDS-PAGE sample buffer, boiled for 5 minutes and then identical quantities of every sample were resolved through SDS-PAGE. Separated proteins were subsequently transferred onto nitrocellulose membrane using semi-dry transfer apparatus. Transfer of proteins were assessed by Ponceau staining, membrane was then washed and blocked with 5% non-fat skimmed milk and followed by incubation with primary antibodies. The dilutions of primary antibodies used were as follows: Ube3a and GAPDH at 1:5000; BDNF and Arc at 1:3000. Blots were further washed, incubated with appropriate secondary antibody followed by detection with ECL reagent.

Immunofluorescence and Golgi staining

Mice were anesthetized by injecting ketamine (100 mg/kg body weight) and Xylazine (10 mg/kg body weight) and then perfused first with only PBS and then by 4% PFA in PBS. After perfusion, brains were isolated and stored in 4% PFA for 24 hours and then immersed into 10, 20 and 30% sucrose solution for 24 hours each at 4°C. Serial brain sections were prepared (20 µm thickness) using cryotome and preserved in PBS having 0.02% sodium azide at 4°C. For immunofluorescence staining, brain sections were first incubated in antigen unmasking solution at 70°C for 40 minutes, were washed with PBS and subjected to endogenous peroxidases blocking (10% H₂O₂, 10% methanol in PBS) for 15 minutes. Sections were next permeabilized by 0.3% triton X-100 for 10 min, blocked with 3% normal goat serum in 0.3% triton X-100 for 2 hours at room temperature followed by incubation with Ube3a antibody (1:500 dilutions) for 12–16 h at 4°C. Sections were washed with PBS and probed with fluorescent-labelled secondary antibodies at room temperature for an hour. After appropriate washing, sections were mounted and imaged using confocal microscope.

For Golgi staining, mice were euthanized, perfused, brain samples were carefully removed and immersed in fixative solution (potassium dichromate and potassium chromate) for 48 hours at 37°C. Fixed brains were then sectioned into thin slices (150µm) using a vibratome (Leica VT1000S). Sections were transferred to glass slides and immersed into the Golgi staining solution containing silver nitrate and potassium chromate and incubated in the dark for several days to allow impregnation of neurons with silver chromate precipitates. The sections were rinsed in distilled water and immersed in the developing solution (equal volumes of 5% ammonia solution and 5% sodium thiosulfate) for 10–15 minutes in the dark. Sections were dehydrated with ethanol and mounted. High-resolution images of stained neurons were captured to visualize dendritic morphology, spine density and spine length. Image analysis software (ImageJ) was used to quantify spine length and spine density. Measurements were taken from multiple neurons within brain region of interest.

Statistical analysis

Experimental data were analyzed using Graphpad Prism software. One or two way analysis of variance followed by Holm-Sidak post-hoc test or Students t-test was used to analyse the data. P < 0.05 was considered statistical significant.

In utero exposure of azadiradione causes reduced body growth and motor dysfunction

In order to understand the impact of azadiradione exposure during development, we treated the timed pregnant mice with two doses of azadiradione (10 mg/kg body weight), one at E12 and another at E14. The details of dosing paradigm, birth of pups, weaning time, and behavioural testing periods are depicted in Fig. 1A. It is important to mention that this dose of azadiradione is well tolerable in adult mice as per our previous studies [11]. We have chosen to inject azadiradione at E12-14, because it is the most commonly used time window to generate chemically-induced model of autism in mice and rats[34]. The litter size, eye opening time and body weight of the newly born pup belongs to vehicle and azadiradione exposed groups were regularly monitored. Azadiradione at this dosing paradigm did not significantly affect the litter size (Fig. 1B). However, injection of azadiradione to the time pregnant mice sequentially at E12, E14 and E16 caused dramatic decrease in litter size. This has led us to avoid the third dose of azadiradione at E16. Eye opening time (ranging from post natal 12.5–13.5 days in both groups) was not significantly affected in comparison with control group when drug was injected at E12 and E14. Interestingly, azadiradione exposure affected the growth of mice as evident from the significantly decreased body weight between postnatal days 10–40 (P10-P40) (Fig. 1C). We further noticed that the azadiradione prenatally exposed mice exhibited increased clasping behaviour, an indication of motor dysfunction (Fig. 1D and 1E). The abnormal clasping behaviour partly improved as mice are grown up to adult stage (Fig. 1E). Similarly, the gait analysis also revealed mild motor dysfunction (as evident from decreased stride length) at P25-30 age group, which was completely recovered when animal reached to P55-60 (Fig. 1G). These findings suggest that in utero exposure of azadiradione leads to mild motor dysfunction at an early developmental stage, which can be restored to normalcy during adult stage.

In utero azadiradione exposure leads to learning and memory deficits

After observing retarded body growth and mild motor dysfunction of the azadiradione prenatally exposed mice, we further assessed their learning and memory function through novel object recognition and 8-arm radial maze test. The novel object recognition test is designed based on the natural inclination of rodents to discover more time on novel object than an accustomed one. The choice to explore novel object is the indication of learning and recognition memory and failure to do so reflect memory impairment. We have observed that azadiradione prenatally exposed mice at P25-30 consumed less time in exploring novel object when compared to age matched vehicle treated controls and therefore having significantly reduced recognition index, which is an indication of learning and memory deficits (Fig. 2A). Interestingly, the reduced visual recognition index in the drug treated animal continues even at P55-65 age group (Fig. 2B). Next we performed radial arm maze test to assess spatial learning and memory. Mice were allowed for 5 days training with all eight arms baited for working memory and four arms baited for reference memory training followed by testing for 5 consecutive days. Working and reference memory errors made by each animal was evaluated. It has been found that azadiradione prenatally exposed mice ended with significantly more working and reference memory errors in 5 days experimental periods as compared to vehicle exposed group not only at P25-35 but also at P55-65 age group (Fig. 2C-F). The findings of radial arm maze as well as novel object recognition tests clearly indicate that the prenatal exposure of azadiradione significantly impairs learning and memory formation and the effect could continue even after 60 days of their birth.

Prenatal exposure of azadiradione leads to altered vocalization and increased anxiety-like behaviour

Mouse ultrasonic vocalizations (USVs) observed in adults and pups, hold substantial communicative value and extensive research has revealed that specific USV patterns appear in various contexts, such as aversive or rewarding situations, as well as during courtship and mating behaviours. Pup isolation calls (PICs) are particularly vital among mouse USVs as they prompt maternal exploration and rescue behaviours and facilitate individual recognition[33]. PICs persist until P15, however with decay in number of calls after P11. Due to their communicative significance, PICs serve as a valuable tool in investigating developmental social communication dysfunction, especially in the context of autism spectrum disorders. Since in utero exposure of azadiradione results is profound learning and memory deficits, we further investigated its effect on the characteristics of neonatal PICs. We found that overall total number of calls produced in the 5 minutes isolation period in the control and treated group were not significantly different on any of the tested days (Fig. 3A). However, on considering the number of calls of different types produced in each day, we found a profound difference in that of the complex type of calls, namely J and H types (Fig. 3B). While there was a mild but significant increase (20%) in the number of S-type calls in treated compared to control, J and H syllables were produced in considerably lesser number (60% for J-type and 54% for H-type) over the 7 days in azadiradione exposed compared to control. Number of noisy syllables did not show any difference. Very few O-type calls were produced. We further analysed the number of calls produced for each type over different days to see if there are specific developmental periods when the alterations are observed. As shown in Fig. 3C-F, J and H types were reduced on P7-P9 and P11 in treated compared to control (P < 0.05). S-type of calls were more in treated compared to control on P6 and P7 (P < 0.01), while N-types were unchanged on all days except a decrease in treated only on the P5. These findings indicate that the complex calls numbers are altered in treated animals in compared to controls and this is overlapped with the onset of hearing[35].

Azadiradione prenatally exposed animals were also subjected to open-field test to asses any anxiety-like behaviour at their age group of P25-30 and P55-60. It was found that azadriadione exposed mice at their age of P25-30 tends to spent more time in the corner area with lesser time in central zone and reduced frequency of core zone entry as compared to control group (Fig. 4, top panel). Azadiradione treated mice also travelled less total distance during the experiment as compared to controls. However, at the age of P55-60, all these parameters in the azadiradione affected animals were nearly similar like control group (Fig. 4 bottom panel).

Azadiradione prenatally exposed mice exhibit increased level of Ube3a and its regulated proteins in their brain

After observing various behavioural deficits in response to prenatal exposure of azadiradione, we next aimed to understand the underlying molecular mechanisms. We first checked the expression of Ube3a as it is shown to be induced by azadiradione. The hippocampal samples were obtained from the vehicle and azadiradione exposed group at P25 and processed for immunoblot analysis using antibody against Ube3a. Figure 5A and B showed that the protein level of Ube3a was significantly higher (more than 3-fold increase) in the hippocampus of azadiradione exposed animals in comparison with vehicle treated group. The transcript level of Ube3a was also significantly increased (Fig. 5C). We further carried out immunofluorescence detection and localization of Ube3a in various brain areas of azadiradione exposed mice along with controls and detected significantly higher level of Ube3a in the hippocampal as well as cortical neurons (Fig. 5D). Ube3a was predominantly localised in the nucleus and drug treatment did not alter its subcellular localisation. This finding is very surprising and suggests that azadiradione prenatal exposure could lead to long lasting expression of Ube3a. We subsequently tested the level of two known downstream targets of Ube3a involved in regulating in synaptic activity and plasticity, namely Arc and BDNF. Immunoblot analysis showed that the level of Arc was markedly down-regulated, while BDNF level was increased (Fig. 6A-C). Transcript level of Arc and BDNF also followed similar trend (Fig. 6D and E). The altered expression of Arc and BDNF at P25 also indirectly supports for the enduring effect of azadiradione on Ube3a expression. Analysis of other known target substrate of Ube3a (because of its ubiquitin ligase activity) could provide further insight in this regards. Nonetheless, increased expression of Ube3a along with Arc and BDNF could potentially alter synaptic function and plasticity in the azadiradione treated animals.

Altered dendritic spine dynamics in azadiradione prenatally exposed mice brain

Since Ube3a regulates neuronal dendritic spine dynamics and abnormal spine density and morphology are one of the common characteristic hallmark of most autism spectrum disorders [36, 37], we further studied the dendritic anomalies in the azadiradione prenatally exposed mice. Brain samples were collected from the azadiradione prenatally exposed mice along with controls at P25 and subjected to Golgi staining procedure. High resolution dendritic images from the Golgi stained pyramidal neurons (both from hippocampal and somatosensory cortex areas) were obtained and analysed for spine density using ImageJ software. As shown in Fig. 7, the numbers of dendritic spine (apical regions) in the hippocampal and cortical pyramidal neurons were significantly increased when azadiradione was exposed before birth. The dendritic spine also seems to be comparatively longer and thinner in azadiradione affected neurons compared to controls. These results suggest that altered dendritic spine dynamics could underlie the observed behavioural deficits in azadiradione prenatally exposed mice.

In the present investigation, we are reporting for the first time that the gestational exposure of azadiradione in mice lead to severe developmental disabilities ranging from impaired physical growth, cognitive, motor and communication deficits and increased anxiety-like behaviours. Most of these abnormalities considerably improved as the animal grown up from early adolescent to adult, though, cognitive deficits significantly persist. While, different parts of the Neem plant in various forms are conventionally used to treat numerous chronic and acute infectious and inflammatory diseases and also as an insecticide, its prenatal effect remains largely unknown[5, 3, 2]. Few reports have indicated congenital malformation of rat foetuses exposed to Neem oil during pregnancy[38, 39]. Accidental Neem oil poisoning in juvenile and adult human beings also results in generalized seizures, loss of consciousness and coma. Ataxia, auditory and visual disturbances also can be seen over time[40–43]. These findings hint for the possible detrimental effect of some of the active ingredients of Neem oil in brain functioning. Neem seeds consist about 35 different types of limonoids and among them azadiradione and gedunin are shown to exhibits neuroprotective activities in animal models of different neurodegenerative disorders, even though, these molecules are cytotoxic at higher doses[9, 11, 44]. It seems azadiradione has completely different effect when exposed during gestational period in compared to adult. Very similar effects can be seen in case of sodium valproate, which is popularly used as an anti-epileptic drug, but its gestational exposure increases the risk of autism[45, 34]. How a molecule like azadiradione could protect degenerated neurons during adult stage, while cause developmental disabilities through gestational exposure? Azadiradione is shown to boost up protein homeostasis and thereby can protect a neuron from the toxic insult of mutant disease proteins (like mutant huntingtin) because of its Ube3a inducing effect[12, 11]. Synaptic dysfunction also could be restored by azadiradione because of the same reason. However, the Ube3a inducing effect of azadiradione during gestational period could lead to detrimental consequences.

The notion behind testing the prenatal effect of azadiradione is based on its ability to induce the expression of Ube3a, which plays a critical role in regulating synaptic activity and plasticity and its expression is precisely regulated during brain development[23, 25, 24, 26, 46]. More importantly, its loss and gain of functions are connected with two different neurodevelopmental disorders[19, 17, 13]. As 2–3 fold increase in the expression/activity of Ube3a results in autism[21, 29, 31], we presumed that prenatal treatment of azadiradione might induce autism-like phenotypes. Indeed, prenatal exposure of azadiradione results in series of behavioural deficits and some of these deficits are analogous to autistic mice. However, many of the behavioural impairments are not the characteristic features of autism, but can be observed in other autism spectrum disorders. There could be various reasons for the diverse behavioural anomalies including multiple targets of the drug, timings of the exposure and the level of induced Ube3a. Series of other behavioural tests related to autism are necessary to draw any further conclusions or to consider whether this small molecule can be used to generate an animal model for autism. Even in valproate induced rodent model of autism, there are reports of wide range of behavioural deficits and it is believed that gestational exposure time as well as dose are important determining factors[45, 47]. Exposure of valproate before and after neural tube closure results is dissimilar behavioural anomalies[47].

Azadiradione prenatally exposed mice also exhibits increased number of dendritic spines that are relatively larger in size (immature morphology) in the pyramidal neurons (hippocampal as well as cortical areas) at P25 and these abnormalities could be linked with the various behavioural deficits observed in these mice. Dendritic spines are very dynamic structure and their numbers, shape and size changes during neural activity associated with learning and memory formation or any sensory experiences[48]. Abnormal dendritic spine density and morphology is the common neuro-morphological feature of autism and autism spectrum disorders[37, 36, 49]. Ube3a-maternal deficient mice (AS mice) display decreased dendritic spine density while mice expressing overactive Ube3a (T485A mutant linked with autism) shows increased number of abnormal spines[31, 30]. Furthermore, Ube3a has been shown to be involved in neural activity-dependent dendritic spine maintenance[46]. These observations indicate that increased dendritic spine density observed in this study could be due to increased expression of Ube3a.

Interestingly, prenatal azadiradione treatment leads to enduring expression of Ube3a in the brain. Exposure of azadiradione to mouse embryos at E12 and E14 stages results in about 3-fold increase in the expression of Ube3a even at P25 meaning the expression persists even about 8 weeks after drug exposure. Altered expression of two crucial Ube3a targets (Arc and BDNF) also detected at P25. Both Arc and BDNF are regulated by Ube3a and plays key role in synaptic activity and plasticity[12, 50–52]. Since Ube3a functions both as ubiquitin ligase and transcriptional co-activator, it is conceivable that many of its target substrates or regulated genes might be affected in the azadiradione affected animals. Although, we do not know the molecular mechanism behind azadiradione-induced up-regulation of Ube3a, it is plausible that the drug might be unsilencing the paternally inherited Ube3a. In that case, azadiradione could be a potent therapeutic molecule to treat AS. The impression is supported by the fact that the known Ube3a unsilencing agent (topoisomerase 1 inhibitor, topotecan), has been shown long lasting effect on Ube3a expression[53]. Any such possibility warrants further investigation.

Altogether, our findings conclude that gestational exposure of azadiradione causes alarming developmental debilities. Ube3a was identified as one of the major cellular targets of azadiradione and its increased and enduring expression could be linked at least in part with the alteration of dendritic spine dynamics and the resulting behavioural deficits.

Acknowledgements: We would like to sincerely thank Ms Rishika Biswas for her technical support.

Funding information: This work was financially sustained by the extramural grant from SERB, Department of Science and Technology (Grant no: CRG/2020/000054) and Department of Biotechnology (BT/PR31122/Med/122/307/2019), Government of India.

Conflict of interest statements: None

Compliance with ethical standards: All experiments were conducted in accordance to the strict guidelines outlined by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forestry, Government of India and were approved by the Institutional Animal Ethics Committee of the Indian Institute of Technology Kharagpur (Protocol number: IE-3/NJ-BS/1.21).

Data availability statement: Data will be made available by the corresponding author on request.

Consent to participate: Not applicable.

Consent for publication: Not applicable.

Author contributions: All authors contributed study conception and design. Experimentations were performed by Sudipta Jana, Sagarika Das, Bhaskar Giri and Raghavendra Archak; data were analysed by Sudipta Jana, Sharba bandyopadhyay and Nihar Ranjan Jana. First draft of the manuscript was written by Nihar Ranjan Jana and all authors read, commented and approved the final manuscript.

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Prenatal exposure of azadiradione leads to developmental disabilities

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Behavioural experiments

Quantitative real-time RT-PCR

Immunoblotting experiments

Immunofluorescence and Golgi staining

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Results

Prenatal exposure of azadiradione leads to altered vocalization and increased anxiety-like behaviour

Altered dendritic spine dynamics in azadiradione prenatally exposed mice brain

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