Biology subjects

Chatterjee, D.

Publications and source records attributed to Chatterjee, D..

69 records · Page 4Linked to original sources

bioRxiv2021

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2

Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinsons disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.

biochemistry↗

bioRxiv2021

Contribution of single mutations to selected SARS-CoV-2 emerging variants Spike antigenicity

Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37{degrees}C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.

microbiology↗

bioRxiv2021

Dynamic behavioral and molecular changes induced by chronic stress exposure in mice

Depression is a leading cause of disabilities around the world, and the underlying mechanisms involved in its pathophysiology are broad and complex. Exposure to chronic stress is a risk factor for developing depressive-symptoms and contributes to cellular and molecular changes precipitating the emergence of symptoms. In the brain, excitatory neurons, inhibitory interneurons and supporting astroglial cells are all sensitive to chronic stress exposure and are known to be impaired in depression. Using an animal model of chronic stress, we assessed the impact of variable durations of chronic stress on the emergence of behavioral deficits and associated molecular changes in the prefrontal cortex (PFC), brain region highly sensitive to stress and impaired in depression. Mice were exposed to up to 35 days of chronic restraint stress and were assessed weekly on behavioral tests measuring anxiety and anhedonia. PFC Protein and RNA levels of specific markers of excitatory, inhibitory synapses and astroglia were quantified using western blot and qPCR, respectively. Correlation and integrative network analyses were used to investigated the impact of chronic stress on the different compartments. Results showed that chronic stress induces anxiety-like behaviors within 7 days, while anhedonia-like behaviors were observed only after 35 days. At the molecular level, alterations of many markers were observed, in particular with longer exposure to chronic stress. Finally, correlation analyses and integrative network analyses revealed that male and female mice react differently to chronic stress exposure and that some markers seem to be more correlated to behaviors deficits in males than in females. Our study demonstrate that chronic induces a dynamic changes that can be observed at the behavioral and molecular levels, and that male and female mice, while exhibiting similar symptoms, have different underlying pathologies.

neuroscience↗

bioRxiv2021

Genetic, Genomic and Biophysical Case Study: Familial 46, XY Sex Reversal due to a Novel Inherited Mutation in Human Testis-Determining Factor SRY

ObjectiveTo describe the clinical, histopathological and molecular features of a novel inherited SRY allele (p.Met64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt. Design, Setting, Participants and Outcome MeasurementsFollowing DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated. ResultsBreast development in P1 and P2 was Tanner II and IV, respectively. Mullerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadablastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Genomic sequencing revealed no candidate mutations in other genes related to sexual differentiation. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancers Enh13 and TESCO. Biophysical studies indicated that the mutant HMG box retains specific DNA binding and DNA bending but with accelerated rate of protein-DNA dissociation. ConclusionThe partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the "the father-daughter paradox"). Steroidal biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

developmental biology↗

bioRxiv2021

Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth

Among CD4+ T-cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier resulting in microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long-term in the gastro-intestinal lymphatic tract, where low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence, however it is unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host-cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T-cells. Depletion of RORC2 inhibited HIV-1 infection, whereas RORC2 overexpression enhanced it. RORC2 was found to promote HIV-1 gene expression. Chromatin immune precipitation revealed that RORC2 binds to the nuclear receptor responsive element (NRRE) in the HIV-1 LTR. In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T-cells from antiretroviral-treated patients. Altogether, these results provide a new explanation as to why Th17 cells are highly susceptible to HIV-1 infection and point to RORC2 as a cell-specific target for HIV-1 therapy. Significance statementHIV-1 infects CD4 T cells and among these, Th17 cells are known to be particularly permissive for virus replication. Infection of Th17 cells is critical for AIDS pathogenesis and viral persistence, however it is not clear why these cells are highly permissive to HIV-1. We found that Th17 cell permissiveness depends on expression of the hormone receptor RORC2, which is the master transcriptional regulator of Th17 cell differentiation. We identify RORC2 as a new, cell-specific host-dependency factor that can be targeted by small molecules. Our results point to RORC2 as a cell-specific target for HIV-1 therapy, an entirely new concept in the field, and suggest HIV-1 might have evolved to exploit RORC2 to promote its own persistence.

microbiology↗

bioRxiv2021

Chronic Stress Alters Astrocyte Morphology in Mouse Prefrontal Cortex

BackgroundNeuromorphological changes are consistently reported in the prefrontal cortex (PFC) of patients with stress-related disorders and in rodent stress models, but the effects of stress on astrocyte morphology and potential link to behavioral deficits are relatively unknown. MethodsTo answer these questions, transgenic mice expressing green fluorescent protein (GFP) under the glial fibrillary acid protein (GFAP) promotor were subjected to 7, 21 or 35 days of chronic restraint stress (CRS). CRS induced behavioral effects on anhedonia- and anxiety-like behaviors were measured using the sucrose intake and the PhenoTyper tests, respectively. PFC GFP+ or GFAP+ cells morphology was assessed using Sholl analysis and associations with behavior were determined using correlation analysis. ResultsCRS-exposed male and female mice displayed anxiety-like behavior at 7, 21 and 35 days and anhedonia-like behavior at 35 days. Analysis of GFAP+ cell morphology revealed significant atrophy of distal processes following 21 and 35 days of CRS. CRS induced similar decreases in intersections at distal radii for GFP+ cells, accompanied by increased proximal processes. In males, the number of intersections at the most distal radius step significantly correlated with anhedonia-like behavior (r=0.622, p<0.05) for GFP+ cells and with behavioral emotionality calculated by z-scoring all behavioral measured deficits (r=-0.667, p<0.05). Similar but not significant correlations were observed in females. No correlation between GFP+ cell atrophy with anxiety-like behavior was found. ConclusionChronic stress exposure induces a progressive atrophy of cortical astroglial cells, potentially contributing to maladaptive neuroplastic and behavioral changes associated with stress-related disorders.

animal behavior and cognition↗

bioRxiv2020

Amyloid fibril-based hydrogels for high-throughput tumor spheroid modeling

Biomaterials mimicking extracellular matrices (ECM) for three-dimensional (3D) cultures have gained immense interest in tumor modeling and in vitro organ development. Here, we introduce versatile, thixotropic amyloid hydrogels as a bio-mimetic ECM scaffold for 3D cell culture as well as high-throughput tumor spheroid formation using a drop cast method. The unique cross-{beta}-sheet structure, sticky surface, and thixotropicity of amyloid hydrogels allow robust cell adhesion, survival, proliferation, and migration, which are essential for 3D tumor modeling with various cancer cell types. The spheroids formed show overexpression of the signature cancer biomarkers and confer higher drug resistance compared to two-dimensional (2D) monolayer cultures. Using breast tumor tissue from mouse xenograft, we showed that these hydrogels support the formation of tumor spheroids with a well-defined necrotic core, cancer-associated gene expression, higher drug resistance, and tumor heterogeneity reminiscent of the original tumor. Altogether, we have developed a rapid and cost-effective platform for generating in vitro cancer models for the screening of anti-cancer therapeutics and developing personalized medicines.

bioengineering↗

bioRxiv2020

Detection And Purification Of Lewy Pathology From Formalin Fixed Primary Human Tissue Using Biotinylation By Antigen Recognition

The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called Lewy pathology is a central phenomenon for the pathogenesis of Parkinsons disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy. Understanding the molecular architecture of Lewy pathology is crucial for understanding disease origins and progression. Here we developed a method to label, extract, and purify molecules from Lewy pathology of formalin fixed PD and DLB brain for blotting and mass spectrometry analysis. Using the biotinylation antibody recognition (BAR) technique, we labeled phosphoserine 129 alpha-synuclein positive pathology and associated molecules with biotin. Formalin crosslinks were then reversed, protein extracted, and pathology associated molecules isolated with streptavidin beads. Results showed superior immunohistochemical staining of Lewy pathology following the BAR protocol when compared to standard avidin biotin complex (ABC) based detection. The enhanced staining was particularly apparent for fibers of the medial forebrain bundle and punctate pathology within the striatum and cortex, which otherwise were weakly labeled or not detected. Subsequent immunoblotting BAR-labeled Lewy pathology extracts revealed the presence of high molecular weight alpha-synuclein, ubiquitin protein conjugates, and phosphoserine 129 alpha-synuclein. Mass spectrometry analysis of BAR-labeled Lewy pathology extracts from PD and DLB patients identified 815 proteins with significant enrichment for many pathways. Notably the most significant KEGG pathway was Parkinsons disease (FDR = 2.48 x 10-26) and GO Cellular compartment was extracellular exosomes (GO Cellular Compartment; FDR = 2.66x 10-34). We used enrichment data to create a functional map of Lewy Pathology from primary disease tissues, which implicated Vesicle Trafficking as the primary disease associated pathway in DLB and PD. In summary, this protocol can be used to enrich for Lewy pathology from formalin fixed human primary tissues, which allows the determination of molecular signatures of Lewy pathology. This technique has broad potential to help understand the phenomenon of Lewy pathology in primary human tissue and animal models.

neuroscience↗

bioRxiv2020

Nucleotide binding, evolutionary insights and interaction partners of the pseudokinase Unc-51-like kinase 4

Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the cofactor is required for structural stability of ULK4. Here we present a high-resolution structure of a ULK4-ATP{gamma}S complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to C. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4 specific activation loop, which stabilizes the C-helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin associated proteins and several active kinases suggesting new roles for ULK4. HighlightsStructure of the ULK4 ATP complex reveals a unique ATP binding mode. Disease associated mutations modulate ATP binding and ULK4 stability Degradation of active site motifs co-occurred in evolution with an ULK4 specific activation loop BioID suggests a role of ULK4 regulating centrosomal and cytoskeletal functions

biochemistry↗

bioRxiv2020

Avid binding by B cells to the Plasmodium circumsporozoite protein repeat suppresses responses to protective subdominant epitopes

Antibodies targeting the NANP/NVDP repeat domain of the Plasmodium falciparum circumsporozoite protein (CSPRepeat) can confer protection against malaria. However, it has also been suggested that this repeat domain exists as a decoy that distracts the immune system from mounting protective responses targeting other domains of CSP. Here we show that B cell responses to the repeat domain are indeed [~]10 fold higher than responses to the N- and C-terminal regions of CSP after sporozoite immunization. We investigated the role of the number of CSPRepeat-specific naive precursor B cells and high avidity binding by B cells in driving the immunodominance of the CSPRepeat. Using adoptive transfer of germline precursors specific for the CSPRepeat, we found that increasing precursor number did indeed increase the responses to the repeat region, but not to the detriment of responses to other epitopes. To investigate the role of avid binding by B cells to the CSPRepeat in driving immunodominance we generated CSP9: a truncated CSP molecule with just 9 NANP repeats. Compared to near full length CSP molecules, CSP9 induced lower BCR signalling in CSPRepeat-specific cells and induced stronger responses to non-repeat epitopes. Finally, we found mice immunized with CSP9 molecules were strongly protected against mosquito bite challenge. Collectively these data demonstrate that the CSPRepeat does function as an immunodominant decoy and that truncated CSP molecules may be a promising avenue for future malaria vaccines. Significance StatementMalaria kills approximately 420,000 individuals each year(1). Our best vaccine, RTS,S/AS01 is based on the circumsporozoite protein that coasts the surface of the parasite. However, this vaccine is only partially protective. Here we show that responses to a repeat region in the circumsporozoite dominate the immune response. However, immunizing with a circumsporozoite protein with a shortened repeat region induces a more diverse immune response, which could be an avenue to make better malaria vaccines.

immunology↗

bioRxiv2019

Genotype Effects and Gene x Environment Interactions on Inter-Individual Licking Received and Licking Provisioning in Female Rat Offspring

In most mammals, mothers exhibit natural variations in care that propagate between generations of female offspring. However, there is limited information on genetic variation that influences this propagation. We assessed early-life maternal care received by individual female rat offspring in relation to genetic polymorphisms linked to dopaminergic activity, maternal care provisioning, and dopaminergic activity in the maternal brain. We also conducted a systematic analysis of other genetic variants potentially related to maternal behavior in our Long-Evans rat population. We found that dopamine receptor 2 (rs107017253) variation interacted with the relationship between early-life maternal care received and dopamine levels in the nucleus accumbens which, in turn, were associated with later-life maternal care provisioning. We also discovered and validated new variants that were predicted by our systematic analysis. Our findings suggest that genetic variation influences the relationship between maternal care received and maternal care provisioning, similar to findings in human populations.

neuroscience↗

bioRxiv2019

Antibody feedback limits the expansion of cognate memory B cells but drives the diversification of vaccine-induced antibody responses

Generating sufficient antibody to block infection is a key challenge for vaccines against malaria. Here we show that antibody titres to a key target, the repeat region of the Plasmodium falciparum circumsporozoite protein (PfCSP), plateaued after two immunizations in a clinical trial of the radiation-attenuated sporozoite vaccine. To understand the mechanisms limiting vaccine responsiveness, we developed Ig-knockin mice with elevated numbers of PfCSP-binding B cells. We determined that recall responses were inhibited by antibody feedback via epitope masking of the immunodominant PfCSP repeat region. Importantly, the amount of antibody that prevents boosting is below the amount of antibody required for protection. Finally, while antibody feedback limited responses to the PfCSP-repeat region in vaccinated volunteers, potentially protective subdominant responses to C-terminal regions did expand with subsequent boosts. These data suggest that antibody feedback drives the diversification of immune responses and that vaccination for malaria will require the targeting of multiple antigens.

immunology↗

bioRxiv2019

Single-cell RNA sequencing of adult Drosophila ovaryidentifies transcriptional programs governing oogenesis

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modelled extensively using the Drosophila ovary. While different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic dataset for the adult Drosophila ovary and connected tissues. This dataset captures the entire transcriptional trajectory of the developing follicle cell population over time. Our findings provide detailed insight into processes such as cell-cycle switching, migration, symmetry breaking, nurse cell engulfment, egg-shell formation, and signaling during corpus luteum formation, marking a newly identified oogenesis-to-ovulation transition. Altogether, these findings provide a broad perspective on oogenesis at a single-cell resolution while revealing new genetic markers and fate-specific transcriptional signatures to facilitate future studies.

developmental biology↗

bioRxiv2019

Liquid-liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation

-Synuclein (-Syn) aggregation and amyloid formation is directly linked with Parkinsons disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of -Syn precedes its aggregation. In particular, in vitro generated -Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate -Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote -Syn LLPS and its subsequent maturation. We further demonstrate -Syn liquid droplet formation in cells, under oxidative stress. These cellular -Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured -Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

neuroscience↗

bioRxiv2019

Loss of one Engrailed1 allele enhances induced α-synucleinopathy

BackgroundParkinsons disease (PD) is a synucleinopathy that has multiple neuropathological characteristics, with nigrostriatal dopamine system degeneration being a core feature. Current models of PD pathology typically fail to recapitulate several attributes of the pathogenic process and neuropathology. We aimed to define the effects of combining a mouse model exhibiting multiple PD-like changes with intrastriatal injections of -synuclein (-syn) pre-formed fibrils (PFFs) aggregates. We employed the heterozygous Engrailed 1 (En1+/-) mouse that features several pathophysiological hallmarks of clinical PD. Objective: To test the hypothesis that the neuropathological changes in the En1+/- mice will promote formation of -syn aggregates following intrastriatal injections of pathogenic human -syn PFFs. Methods: We unilaterally injected PFFs into the striata of 1 month-old En1+/- and control wild-type mice and euthanized animals at 3 months for post-mortem analysis. Results: Using immunohistochemistry and unbiased stereology, we established that PFF-injected En1+/- mice exhibited a near-threefold increase in pS129--syn-positive neurons in the substantia nigra compared to PFF-injected wild-type mice. The PFF-injected En1+/- mice also displayed significant increases in pS129--syn-positive neurons in the amygdala and ventral tegmental area; regions of known PD pathology with projections to the striatum. Additionally, we observed amplified pS129--syn-positive aggregation in En1+/- mice in multiple cortical regions. Conclusions: Following intrastriatal injection of PFFs, absence of an En1 allele leads to additional aggregation of pathological -syn, potentially due to En1-loss mediated nigrostriatal impairment. We propose that further development of this double-hit model could be predictive of pre-clinical therapeutic potential and success for PD than existing mouse models.

neuroscience↗