@article {3346, title = {Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis.}, journal = {Mol Neurobiol}, volume = {59}, year = {2022}, month = {2022 Dec}, pages = {7370-7392}, abstract = {

The Fragile-X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation of mRNAs essential for synaptic development and plasticity. FMRP interacts with a specific set of mRNAs, aids in their microtubule-dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of FMRP{\textquoteright}s domains in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. We report that the C-terminus domain of FMRP is sufficient to bind to ribosomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated neuronal translation repression. However, dendritic distribution of FMRP and its microtubule association is favored by the synergistic combination of FMRP domains rather than individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome association. This is a fundamental mechanism governing the size and number of FMRP puncta that contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchical contribution of FMRP{\textquoteright}s domains in translation regulation.

}, keywords = {Fragile X Mental Retardation Protein, Fragile X Syndrome, Humans, Microtubules, Neurons, Protein Biosynthesis, Ribosomes, RNA, Messenger}, issn = {1559-1182}, doi = {10.1007/s12035-022-03049-1}, author = {D{\textquoteright}Souza, Michelle Ninochka and Ramakrishna, Sarayu and Radhakrishna, Bindushree K and Jhaveri, Vishwaja and Ravindran, Sreenath and Yeramala, Lahari and Nair, Deepak and Palakodeti, Dasaradhi and Muddashetty, Ravi S} } @article {3339, title = {NMDAR mediated dynamic changes in mA inversely correlates with neuronal translation.}, journal = {Sci Rep}, volume = {12}, year = {2022}, month = {2022 07 05}, pages = {11317}, abstract = {

Epitranscriptome modifications are crucial in translation regulation and essential for maintaining cellular homeostasis. N6 methyladenosine (mA) is one of the most abundant and well-conserved epitranscriptome modifications, which is known to play a pivotal role in diverse aspects of neuronal functions. However, the role of mA modifications with respect to activity-mediated translation regulation and synaptic plasticity has not been studied. Here, we investigated the role of mA modification in response to NMDAR stimulation. We have consistently observed that 5\ min NMDAR stimulation causes an increase in eEF2 phosphorylation. Correspondingly, NMDAR stimulation caused a significant increase in the mA signal at 5\ min time point, correlating with the global translation inhibition. The NMDAR induced increase in the mA signal is accompanied by the redistribution of the mA marked RNAs from translating to the non-translating pool of ribosomes. The increased mA levels are well correlated with the reduced FTO levels observed on NMDAR stimulation. Additionally, we show that inhibition of FTO prevents NMDAR mediated changes in mA levels. Overall, our results establish RNA-based molecular readout which corelates with the NMDAR-dependent translation regulation which helps in understanding changes in protein synthesis.

}, keywords = {Adenosine, Neurons, Phosphorylation, Receptors, N-Methyl-D-Aspartate, RNA}, issn = {2045-2322}, doi = {10.1038/s41598-022-14798-3}, author = {Gowda, Naveen Kumar Chandappa and Nawalpuri, Bharti and Ramakrishna, Sarayu and Jhaveri, Vishwaja and Muddashetty, Ravi S} } @article {2328, title = {APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis.}, journal = {J Neurosci}, volume = {41}, year = {2021}, month = {2021 Oct 20}, pages = {8686-8709}, abstract = {

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer{\textquoteright}s disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis. Defective protein synthesis has been shown as an early defect in familial Alzheimer{\textquoteright}s disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.0435-21.2021}, author = {Ramakrishna, Sarayu and Jhaveri, Vishwaja and Konings, Sabine C and Nawalpuri, Bharti and Chakraborty, Sumita and Holst, Bj{\o}rn and Schmid, Benjamin and Gouras, Gunnar K and Freude, Kristine K and Muddashetty, Ravi S} } @article {2362, title = {Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3.}, journal = {Stem Cell Reports}, volume = {16}, year = {2021}, month = {2021 Nov 09}, pages = {2736-2751}, abstract = {

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

}, issn = {2213-6711}, doi = {10.1016/j.stemcr.2021.09.013}, author = {Chandrasekaran, Abinaya and Dittlau, Katarina Stoklund and Corsi, Giulia I and Haukedal, Henriette and Doncheva, Nadezhda T and Ramakrishna, Sarayu and Ambardar, Sheetal and Salcedo, Claudia and Schmidt, Sissel I and Zhang, Yu and Cirera, Susanna and Pihl, Maria and Schmid, Benjamin and Nielsen, Troels Tolstrup and Nielsen, J{\o}rgen E and Kolko, Miriam and Kobol{\'a}k, Julianna and Dinny{\'e}s, Andr{\'a}s and Hyttel, Poul and Palakodeti, Dasaradhi and Gorodkin, Jan and Muddashetty, Ravi S and Meyer, Morten and Aldana, Blanca I and Freude, Kristine K} } @article {2329, title = {Distinct temporal expression of the GW182 paralog TNRC6A in neurons regulates dendritic arborization.}, journal = {J Cell Sci}, volume = {134}, year = {2021}, month = {2021 Aug 15}, abstract = {

Precise development of the dendritic architecture is a critical determinant of mature neuronal circuitry. MicroRNA (miRNA)-mediated regulation of protein synthesis plays a crucial role in dendritic morphogenesis, but the role of miRNA-induced silencing complex (miRISC) protein components in this process is less studied. Here, we show an important role of a key miRISC protein, the GW182 paralog TNRC6A, in the regulation of dendritic growth. We identified a distinct brain region-specific spatiotemporal expression pattern of GW182 during rat postnatal development. We found that the window of peak GW182 expression coincides with the period of extensive dendritic growth, both in the hippocampus and cerebellum. Perturbation of GW182 function during a specific temporal window resulted in reduced dendritic growth of cultured hippocampal neurons. Mechanistically, we show that GW182 modulates dendritic growth by regulating global somatodendritic translation and actin cytoskeletal dynamics of developing neurons. Furthermore, we found that GW182 affects dendritic architecture by regulating the expression of actin modulator LIMK1. Taken together, our data reveal a previously undescribed neurodevelopmental expression pattern of GW182 and its role in dendritic morphogenesis, which involves both translational control and actin cytoskeletal rearrangement. This article has an associated First Person interview with the first author of the paper.

}, issn = {1477-9137}, doi = {10.1242/jcs.258465}, author = {Nawalpuri, Bharti and Sharma, Arpita and Chattarji, Sumantra and Muddashetty, Ravi S} } @article {2067, title = {Distinct regulation of bioenergetics and translation by group I mGluR and NMDAR.}, journal = {EMBO Rep}, year = {2020}, month = {2020 Apr 29}, pages = {e48037}, abstract = {

Neuronal activity is responsible for the high energy consumption in the brain. However, the cellular mechanisms draining ATP upon the arrival of a stimulus are yet to be explored systematically at the post-synapse. Here, we provide evidence that a significant fraction of ATP is consumed upon glutamate stimulation to energize mGluR-induced protein synthesis. We find that both mGluR and NMDAR alter protein synthesis and ATP consumption with distinct kinetics at the synaptic-dendritic compartments. While mGluR activation leads to a rapid and sustained reduction in neuronal ATP levels, NMDAR activation has no immediate impact on the same. ATP consumption correlates inversely with the kinetics of protein synthesis for both receptors. We observe a persistent elevation in protein synthesis within 5\ minutes of mGluR activation and a robust inhibition of the same within 2\ minutes of NMDAR activation, assessed by the phosphorylation status of eEF2 and metabolic labeling. However, a delayed protein synthesis-dependent ATP expenditure ensues after 15\ minutes of NMDAR stimulation. We identify a central role for AMPK in the correlation between protein synthesis and ATP consumption. AMPK is dephosphorylated and inhibited upon mGluR activation, while it is phosphorylated upon NMDAR activation. Perturbing AMPK activity disrupts receptor-specific modulations of eEF2 phosphorylation and protein synthesis. Our observations, therefore, demonstrate that the regulation of the AMPK-eEF2 signaling axis by glutamate receptors alters neuronal protein synthesis and bioenergetics.

}, issn = {1469-3178}, doi = {10.15252/embr.201948037}, author = {Ghosh Dastidar, Sudhriti and Das Sharma, Shreya and Chakraborty, Sumita and Chattarji, Sumantra and Bhattacharya, Aditi and Muddashetty, Ravi S} } @article {2059, title = {The Role of Dynamic miRISC During Neuronal Development.}, journal = {Front Mol Biosci}, volume = {7}, year = {2020}, month = {2020}, pages = {8}, abstract = {

Activity-dependent protein synthesis plays an important role during neuronal development by fine-tuning the formation and function of neuronal circuits. Recent studies have shown that miRNAs are integral to this regulation because of their ability to control protein synthesis in a rapid, specific and potentially reversible manner. miRNA mediated regulation is a multistep process that involves inhibition of translation before degradation of targeted mRNA, which provides the possibility to store and reverse the inhibition at multiple stages. This flexibility is primarily thought to be derived from the composition of miRNA induced silencing complex (miRISC). AGO2 is likely the only obligatory component of miRISC, while multiple RBPs are shown to be associated with this core miRISC to form diverse miRISC complexes. The formation of these heterogeneous miRISC complexes is intricately regulated by various extracellular signals and cell-specific contexts. In this review, we discuss the composition of miRISC and its functions during neuronal development. Neurodevelopment is guided by both internal programs and external cues. Neuronal activity and external signals play an important role in the formation and refining of the neuronal network. miRISC composition and diversity have a critical role at distinct stages of neurodevelopment. Even though there is a good amount of literature available on the role of miRNAs mediated regulation of neuronal development, surprisingly the role of miRISC composition and its functional dynamics in neuronal development is not much discussed. In this article, we review the available literature on the heterogeneity of the neuronal miRISC composition and how this may influence translation regulation in the context of neuronal development.

}, issn = {2296-889X}, doi = {10.3389/fmolb.2020.00008}, author = {Nawalpuri, Bharti and Ravindran, Sreenath and Muddashetty, Ravi S} } @article {1646, title = {BDNF Induced Translation of Limk1 in Developing Neurons Regulates Dendrite Growth by Fine-Tuning Cofilin1 Activity.}, journal = {Front Mol Neurosci}, volume = {12}, year = {2019}, month = {2019}, pages = {64}, abstract = {

Dendritic growth and branching are highly regulated processes and are essential for establishing proper neuronal connectivity. There is a critical phase of early dendrite development when these are heavily regulated by external cues such as trophic factors. Brain-derived neurotrophic factor (BDNF) is a major trophic factor known to enhance dendrite growth in cortical neurons, but the molecular underpinnings of this response are not completely understood. We have identified that BDNF induced translational regulation is an important mechanism governing dendrite development in cultured rat cortical neurons. We show that BDNF treatment for 1 h in young neurons leads to translational up-regulation of an important actin regulatory protein LIM domain kinase 1 (Limk1), increasing its level locally in the dendrites. Limk1 is a member of serine/threonine (Ser/Thr) family kinases downstream of the Rho-GTPase pathway. BDNF induced increase in Limk1 levels leads to increased phosphorylation of its target protein cofilin1. We observed that these changes are maintained for long durations of up to 48 h and are mediating increase in number of primary dendrites and total dendrite length. Thus, we show that BDNF induced protein synthesis leads to fine-tuning of the actin cytoskeletal reassembly and thereby mediate dendrite development.

}, issn = {1662-5099}, doi = {10.3389/fnmol.2019.00064}, author = {Ravindran, Sreenath and Nalavadi, Vijayalaxmi C and Muddashetty, Ravi S} } @article {1982, title = {Differential Regulation of Translation by FMRP Modulates eEF2 Mediated Response on NMDAR Activity.}, journal = {Front Mol Neurosci}, volume = {12}, year = {2019}, month = {2019}, pages = {97}, abstract = {

SYNGAP1, a Synaptic Ras-GTPase activating protein, regulates synapse maturation during a critical developmental window. Heterozygous mutation in () has been shown to cause Intellectual Disability (ID) in children. Recent studies have provided evidence for altered neuronal protein synthesis in a mouse model of . However, the molecular mechanism behind the same is unclear. Here, we report the reduced expression of a known translation regulator, FMRP, during a specific developmental period in mice. Our results demonstrate that FMRP interacts with and regulates the translation of mRNA. We further show reduced translation leads to decreased FMRP level during development in which results in an increase in translation. These developmental changes are reflected in the altered response of eEF2 phosphorylation downstream of NMDA Receptor (NMDAR)-mediated signaling. In this study, we propose a cross-talk between FMRP and SYNGAP1 mediated signaling which can also explain the compensatory effect of impaired signaling observed in mice.

}, issn = {1662-5099}, doi = {10.3389/fnmol.2019.00097}, author = {Paul, Abhik and Nawalpuri, Bharti and Shah, Devanshi and Sateesh, Shruthi and Muddashetty, Ravi S and Clement, James P} } @article {1739, title = {Effect of early maternal separation stress on attention, spatial learning and social interaction behaviour.}, journal = {Exp Brain Res}, volume = {237}, year = {2019}, month = {2019 Aug}, pages = {1993-2010}, abstract = {

Early life stress is known to influence affective and cognitive functions in later life but comprehensive explanation for the impact of early life stress on attentional functions, behavioural control and social behaviour is inadequate. The early life stress was induced by exposing rat pups to 6\ h of maternal separation and isolation (MS) stress from postnatal days 4-14 i.e. during SHRP period. The long-term impact of MS in these rats was evaluated by assessing anxiety, sociability, social preference, spatial learning and memory along with a detailed evaluation of attentional functions during young adulthood period. Adult male MS rats showed increased anxiety-like behaviour, impaired flexibility in social interactions, and increased reward-seeking behaviour. MS rats also showed faster spatial learning in the partially baited radial arm maze and exhibited moderately enhanced sustained attention in the 5-choice serial reaction time task (5CSRTT). These results suggest that early MS has both positive and negative consequences in adulthood. Increased cognitive ability in MS rats, as evidenced by the improved sustained attention and spatial learning and memory, is usually advantageous and indicates positive influences of early stressors that might lead to the development of resilience and enhanced compensatory mechanisms later in adulthood. MS stress has compromised flexibility in social behaviour that promotes solitary lifestyle and social isolation. Heightened reward-seeking behaviour, as shown by the MS rats, could be a predisposing factor for substance abuse and addiction. Thus, our study highlights the crucial and differential impact of early life challenges on behaviour during adulthood and suggests that the positive aspects could be an asset that may be utilized to suppress the negative effects of early life stress in adulthood.

}, issn = {1432-1106}, doi = {10.1007/s00221-019-05567-2}, author = {Kambali, Maltesh Y and Anshu, Kumari and Kutty, Bindu M and Muddashetty, Ravi S and Laxmi, T Rao} } @article {1611, title = {Emerging Role of microRNAs in Dementia.}, journal = {J Mol Biol}, year = {2019}, month = {2019 Feb 07}, abstract = {

MicroRNAs are small non-coding RNAs regulating mRNA translation. They play a crucial role in regulating homeostasis in neurons, especially in regulating local and stimulation dependent protein synthesis. Since activity-mediated protein synthesis in neurons is critical for memory and cognition, microRNAs have become key players in modulating these processes. Dementia is a broad term used for symptoms involving decline of memory and cognition. Several studies have implicated the dysregulation of microRNAs in many brain diseases like neurodegenerative diseases, neurodevelopmental disorders, brain injuries and dementia. In this review, we give an overview of microRNA-mediated regulation of proteins and cellular processes affected in dementia pathology, hence illustrating the importance of microRNAs in normal functioning. We also focus on a relatively less explored area in dementia pathology-the importance of activity-mediated protein synthesis at the synapse and the role of microRNAs in modulating this. Overall, this review will be helpful in looking at the significance of microRNAs in dementia from the perspective of defective regulation of protein synthesis and synaptic dysfunction.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2019.01.046}, author = {Ramakrishna, Sarayu and Muddashetty, Ravi S} } @article {1841, title = {Generation of a FMR1 homozygous knockout human embryonic stem cell line (WAe009-A-16) by CRISPR/Cas9 editing.}, journal = {Stem Cell Res}, volume = {39}, year = {2019}, month = {2019 Aug}, pages = {101494}, abstract = {

Mutations in FMR1 gene is the cause of Fragile X Syndrome (FXS) leading inherited cause of intellectual disability and autism spectrum disorders. FMR1 gene encodes Fragile X Mental Retardation Protein (FMRP) which is a RNA binding protein and play important role in synaptic plasticity and translational regulation in neurons. We have generated a homozygous FMR1 knockout (FMR1-KO) hESC line using CRISPR/Cas9 based genome editing. It created a homozygous 280 nucleotide deletion at exon1, removing the start codon. This FMR1-KO cell line maintains stem cell like morphology, pluripotency, normal karyotype and ability to in-vitro differentiation.

}, issn = {1876-7753}, doi = {10.1016/j.scr.2019.101494}, author = {Giri, Subhajit and Purushottam, Meera and Viswanath, Biju and Muddashetty, Ravi S} } @article {1744, title = {NMDAR mediated translation at the synapse is regulated by MOV10 and FMRP.}, journal = {Mol Brain}, volume = {12}, year = {2019}, month = {2019 Jul 10}, pages = {65}, abstract = {

Protein synthesis is crucial for maintaining synaptic plasticity and synaptic signalling. Here we have attempted to understand the role of RNA binding proteins, Fragile X Mental Retardation Protein (FMRP) and Moloney Leukemia Virus 10 (MOV10) protein in N-Methyl-D-Aspartate Receptor (NMDAR) mediated translation regulation. We show that FMRP is required for translation downstream of NMDAR stimulation and MOV10 is the key specificity factor in this process. In rat cortical synaptoneurosomes, MOV10 in association with FMRP and Argonaute 2 (AGO2) forms the inhibitory complex on a subset of NMDAR responsive mRNAs. On NMDAR stimulation, MOV10 dissociates from AGO2 and promotes the translation of its target mRNAs. FMRP is required to form MOV10-AGO2 inhibitory complex and to promote translation of MOV10 associated mRNAs. Phosphorylation of FMRP appears to be the potential switch for NMDAR mediated translation and in the absence of FMRP, the distinct translation response to NMDAR\ stimulation is lost. Thus, FMRP and MOV10 have an important regulatory role in NMDAR mediated translation at the synapse.

}, issn = {1756-6606}, doi = {10.1186/s13041-019-0473-0}, author = {Kute, Preeti Madhav and Ramakrishna, Sarayu and Neelagandan, Nagammal and Chattarji, Sumantra and Muddashetty, Ravi S} } @article {1598, title = {FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation.}, journal = {iScience}, volume = {9}, year = {2018}, month = {2018 Nov 30}, pages = {399-411}, abstract = {

FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2{\textquoteright}O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2{\textquoteright}O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

}, issn = {2589-0042}, doi = {10.1016/j.isci.2018.11.007}, author = {D{\textquoteright}Souza, Michelle Ninochka and Gowda, Naveen Kumar Chandappa and Tiwari, Vishal and Babu, Rosana Ottakandathil and Anand, Praveen and Dastidar, Sudhriti Ghosh and Singh, Randhir and James, Owen G and Selvaraj, Bhuvaneish and Pal, Rakhi and Ramesh, Arati and Chattarji, Sumantra and Chandran, Siddharthan and Gulyani, Akash and Palakodeti, Dasaradhi and Muddashetty, Ravi S} }