TY - JOUR T1 - FMRP protects the lung from xenobiotic stress by facilitating the Integrated Stress Response. JF - J Cell Sci Y1 - 2022 A1 - Basu, Deblina Sain A1 - Bhavsar, Rital A1 - Gulami, Imtiyaz A1 - Chavda, Saraswati A1 - Lingamallu, Sai Manoz A1 - Muddashetty, Ravi A1 - Veeranna, Chandrakanth A1 - Chattarji, Sumantra A1 - Thimmulappa, Rajesh A1 - Bhattacharya, Aditi A1 - Guha, Arjun AB -

Stress response pathways protect the lung from the damaging effects of environmental toxicants. Here we investigate the role of the Fragile X Mental Retardation Protein (FMRP), a multifunctional protein implicated in stress responses, in the lung. We report that FMRP is expressed in murine and human lungs, in the airways and more broadly. Analysis of airway stress responses in mice and in a murine cell line ex vivo, using the well-established Naphthalene (Nap) injury model, reveals that FMRP-deficient cells exhibit increased expression of markers of oxidative and genotoxic stress and increased cell death. Further inquiry shows that FMRP-deficient cells fail to actuate the Integrated Stress Response Pathway (ISR) and upregulate the transcription factor ATF4. Knockdown of ATF4 expression phenocopies the loss of FMRP. We extend our analysis of the role of FMRP to human bronchial BEAS-2B cells, using a 9, 10-Phenanthrenequinone air pollutant model, to find FMRP-deficient BEAS-2B also fail to actuate the ISR and exhibit greater susceptibility. Taken together, our data suggest that FMRP has a conserved role in protecting the airways by facilitating the ISR.

ER - TY - JOUR T1 - Distinct regulation of bioenergetics and translation by group I mGluR and NMDAR. JF - EMBO Rep Y1 - 2020 A1 - Ghosh Dastidar, Sudhriti A1 - Das Sharma, Shreya A1 - Chakraborty, Sumita A1 - Chattarji, Sumantra A1 - Bhattacharya, Aditi A1 - Muddashetty, Ravi S AB -

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.

ER - TY - JOUR T1 - Dopamine requires unique residues to signal via the serotonin 2A receptor. JF - Neuroscience Y1 - 2020 A1 - Soman, Shuchita A1 - Bhattacharya, Aditi A1 - Panicker, Mitradas M AB -

Serotonin is an important neurotransmitter and neuromodulator. Disruption of the serotonergic system has been implicated in various psychiatric disorders such as schizophrenia and bipolar disorder. Most of the drugs targeting these neurotransmitter systems are classified primarily as agonists or inverse agonists/antagonists, with their described function being limited to activating the canonical signaling pathway(s), or inhibiting the pathway(s) respectively. Previous work with the human 5-HT has shown the receptor to be activated by dopamine, also an endogenous ligand. Dopamine is the cognate ligand of the dopaminergic system, which significantly overlaps with the serotonergic system in the brain. The two systems innervate many of the same brain areas, and the central serotonergic systems also regulate dopamine functions. Our aim was to investigate the downstream signaling set up by the receptor on being activated by dopamine. We show that dopamine is a functionally selective ligand at 5-HT and have examined dopamine as a ligand with respect to some receptor-dependent phenotypes. Our results show that dopamine acts as an agonist at the human serotonin 2A receptor and brings about its activation and internalization. Using in vitro assays, we have established differences in the signaling pathways set up by dopamine as compared to serotonin. Using site-specific mutagenesis we have identified residues important for this functional selectivity, shown by dopamine at this receptor. Our identification of specific residues important in the functional selectivity of dopamine at 5-HT could have far reaching implications for the field of GPCR signaling and drug-design. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

VL - 439 ER - TY - JOUR T1 - N-terminal variant Asp14Asn of the human p70 S6 Kinase 1 enhances translational signaling causing different effects in developing and mature neuronal cells. JF - Neurobiol Learn Mem Y1 - 2020 A1 - Venkatasubramani, Janani Priya A1 - Subramanyam, Prakash A1 - Pal, Rakhi A1 - Reddy, Bharath K A1 - Srinivasan, Durga Jeyalakshmi A1 - Chattarji, Sumantra A1 - Iossifov, Ivan A1 - Klann, Eric A1 - Bhattacharya, Aditi AB -

The ribosomal p70 S6 Kinase 1 (S6K1) has been implicated in the etiology of complex neurological diseases including autism, depression and dementia. Though no major gene disruption has been reported in humans in RPS6KB1, single nucleotide variants (SNVs) causing missense mutations have been identified, which have not been assessed for their impact on protein function. These S6K1 mutations have the potential to influence disease progression and treatment response. We mined the Simon Simplex Collection (SSC) and SPARK autism database to find inherited SNVs in S6K1 and characterized the effect of two missense SNVs, Asp14Asn (allele frequency = 0.03282%) and Glu44Gln (allele frequency = 0.0008244%), on S6K1 function in HEK293, human ES cells and primary neurons. Expressing Asp14Asn in HEK293 cells resulted in increased basal phosphorylation of downstream targets of S6K1 and increased de novo translation. This variant also showed blunted response to the specific S6K1 inhibitor, FS-115. In human embryonic cell line Shef4, Asp14Asn enhanced spontaneous neural fate specification in the absence of differentiating growth factors. In addition to enhanced translation, neurons expressing Asp14Asn exhibited impaired dendritic arborization and increased levels of phosphorylated ERK 1/2. Finally, in the SSC families tracked, Asp14Asn segregated with lower IQ scores when found in the autistic individual rather than the unaffected sibling. The Glu44Gln mutation showed a milder, but opposite phenotype in HEK cells as compared to Asp14Asn. Although the Glu44Gln mutation displayed increased neuronal translation, it had no impact on neuronal morphology. Our results provide the first characterization of naturally occurring human S6K1 variants on cognitive phenotype, neuronal morphology and maturation, underscoring again the importance of translation control in neural development and plasticity.

VL - 171 ER - TY - JOUR T1 - Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition. JF - J Proteome Res Y1 - 2020 A1 - Bowling, Heather L A1 - Kasper, Amanda A1 - Patole, Chhaya A1 - Venkatasubramani, Janani Priya A1 - Leventer, Sarah Parker A1 - Carmody, Erin A1 - Sharp, Kevin A1 - Berry-Kravis, Elizabeth A1 - Kirshenbaum, Kent A1 - Klann, Eric A1 - Bhattacharya, Aditi AB -

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

VL - 19 IS - 9 ER - TY - JOUR T1 - Altered steady state and activity-dependent de novo protein expression in fragile X syndrome. JF - Nat Commun Y1 - 2019 A1 - Bowling, Heather A1 - Bhattacharya, Aditi A1 - Zhang, Guoan A1 - Alam, Danyal A1 - Lebowitz, Joseph Z A1 - Bohm-Levine, Nathaniel A1 - Lin, Derek A1 - Singha, Priyangvada A1 - Mamcarz, Maggie A1 - Puckett, Rosemary A1 - Zhou, Lili A1 - Aryal, Sameer A1 - Sharp, Kevin A1 - Kirshenbaum, Kent A1 - Berry-Kravis, Elizabeth A1 - Neubert, Thomas A A1 - Klann, Eric AB -

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase 1 and Ras, also are expressed in the blood of FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.

VL - 10 IS - 1 ER - TY - JOUR T1 - Hippocampal and amygdalar cell-specific translation is similar soon after stress but diverge over time. JF - Hippocampus Y1 - 2018 A1 - Madan, Jesvin S A1 - Gupta, Kanika A1 - Chattarji, Sumantra A1 - Bhattacharya, Aditi AB -

Stress is known to cause contrasting patterns of morphological and physiological plasticity in the hippocampus and amygdala. An obligatory cellular process underlying such neural changes is de novo translation and alterations in protein expression. Yet the nature of the translational response to stress in neurons remains largely unexplored. Even less is known about how glia are affected. Using a click-chemistry-based method to label the de novo proteome in live brain slices, we monitored translation in neurons and astrocytes of the basolateral amygdala (BLA) and dorsal hippocampal area CA3 (dCA3) in rats at different time-points after a single 2-hr exposure to immobilization stress. We observed enhancements in neuronal translation in both brain regions 1 hour after stress. This initial increase persisted in the BLA up to 10 days afterwards. In contrast, dCA3 neuronal translation gradually decreased to below control levels 10 days later. Translation profiles of dCA3 astrocytes followed timelines similar to neurons, but in BLA astrocytes translation peaked 1 day later and remained elevated 10 days later. Together our results demonstrate that stress causes an immediate upregulation of protein synthesis in both amygdalar and hippocampal neurons and astrocytes. However, these two areas eventually exhibit opposite temporal profiles of protein expression well after the end of stress. These findings identify new metrics of stress-induced plasticity at the level of cell-type specific proteomic landscape that may provide important insights into the molecular basis of the divergent temporal effects of stress across brain regions and biological scales.

VL - 28 IS - 6 ER -