Biology subjects

Santhakumar, V.

Publications and source records attributed to Santhakumar, V..

32 records · Page 2Linked to original sources

bioRxiv2023

Probing the DCAF12 interactions with MAGEA3 and CCT5 C-terminal degrons

DCAF12 is the substrate recognition component of the CRL4 E3 ligase complex that can recognize C-terminal double-glutamic acid degrons to promote degradation of its cognate substrates via the ubiquitin proteasome system. MAGEA3 and CCT5 proteins were reported to be cellular targets of DCAF12. To further characterize the DCAF12 interactions with both MAGEA3 and CCT5, we developed a suite of biophysical and a proximity-based cellular NanoBRET assays showing that both MAGEA3 and CCT5 C-terminal degron peptides interact with DCAF12 in nanomolar affinity in vitro and in cells. Furthermore, we report here the 3.17 [A] cryo-EM structure of DCAF12-DDB1-MAGEA3 complex revealing the key DCAF12 residues involved in C-terminal degron recognition and binding. Our study provides new tools and resources to enable the discovery of small molecule handles targeting the WDR domain of DCAF12 for future PROTAC design and development.

biochemistry↗

bioRxiv2023

Early Deficits in Dentate Circuit and Behavioral Pattern Separation after Concussive Brain Injury

Traumatic brain injury leads to cellular and circuit changes in the dentate gyrus, a gateway to hippocampal information processing. Intrinsic granule cell firing properties and strong feedback inhibition in the dentate are proposed as critical to its ability to generate unique representation of similar inputs by a process known as pattern separation. Here we evaluate the impact of brain injury on cellular decorrelation of temporally patterned inputs in slices and behavioral discrimination of spatial locations in vivo one week after concussive lateral fluid percussion injury (FPI) in mice. Despite posttraumatic increases in perforant path evoked excitatory drive to granule cells and enhanced {Delta}FosB labeling, indicating sustained increase in excitability, the reliability of granule cell spiking was not compromised after FPI. Although granule cells continued to effectively decorrelate output spike trains recorded in response to similar temporally patterned input sets after FPI, their ability to decorrelate highly similar input patterns was reduced. In parallel, encoding of similar spatial locations in a novel object location task that involves the dentate inhibitory circuits was impaired one week after FPI. Injury induced changes in pattern separation were accompanied by loss of somatostatin expressing inhibitory neurons in the hilus. Together, these data suggest that the early posttraumatic changes in the dentate circuit undermine dentate circuit decorrelation of temporal input patterns as well as behavioral discrimination of similar spatial locations, both of which could contribute to deficits in episodic memory.

neuroscience↗

bioRxiv2023

Probing the mechanism of Cbl-b inhibition by a small-molecule inhibitor

Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment, and Nx-1607, is in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirmed potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing compound interaction with both the TKBD and LHR, but not the ring domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.

immunology↗

bioRxiv2023

Discovery and characterization of a chemical probe targeting the zinc-finger ubiquitin-binding domain of HDAC6

Histone deacetylase 6 (HDAC6) inhibition is an attractive strategy for treating numerous cancers, and HDAC6 catalytic inhibitors are currently in clinical trials. The HDAC6 zinc-finger ubiquitin-binding domain (UBD) binds free C-terminal diglycine motifs of unanchored ubiquitin polymer chains and protein aggregates, playing an important role in autophagy and aggresome assembly. However, targeting this domain with small molecule antagonists remains an underdeveloped avenue of HDAC6-focused drug discovery. We report SGC-UBD253 (25), a chemical probe potently targeting HDAC6-UBD in vitro with selectivity over nine other UBDs, except for weak USP16 binding. In cells, 25 is an effective antagonist of HDAC6-UBD at 1 {micro}M, with marked proteome-wide selectivity. We identified SGC-UBD253N (32), a methylated derivative of 25 which is 300-fold less active, serving as a negative control. Together, 25 and 32 could enable further exploration of the biological function of the HDAC6 UBD and investigation of the therapeutic potential of targeting this domain.

pharmacology and toxicology↗

bioRxiv2021

Long-term effects of moderate concussive brain injury during adolescence on synaptic and tonic GABA currents in dentate projection neurons.

Progressive physiological changes in the hippocampal dentate gyrus circuits following traumatic brain injury contribute to temporal evolution of neurological sequelae. Although early posttraumatic changes in dentate synaptic and extrasynaptic GABA currents have been reported, whether they evolve over time and remain distinct between the two projection neuron classes, granule cells and semilunar granule cells, has not been evaluated. We examined changes in tonic GABA currents and spontaneous inhibitory postsynaptic currents (sIPSCs) and in dentate projection neurons one and three month after moderate concussive fluid percussion injury (FPI) in adolescent rats. Granule cell tonic GABA current amplitude remained elevated up to one month after FPI, but decreased to levels comparable to age-matched controls by three months postinjury. Granule cell sIPSC frequency, which we previously reported to be increased one week after FPI, remained higher than in age-matched controls at one month and was significantly reduced three months after FPI. In contrast to the early decrease, tonic GABA current amplitude and sIPSC frequency in semilunar granule cell was not different from controls three months after FPI. The switch in granule cell inhibitory inputs from early increase to subsequent decrease could contribute to the delayed emergence of cognitive deficits and seizure susceptibility after brain injury.

neuroscience↗

bioRxiv2021

Reduced Hippocampal Inhibition and Enhanced Autism-Epilepsy Comorbidity in Mice Lacking Neuropilin 2

The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2 deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2-/- mice have reduced number of parvalbumin, somatostatin and Neuropeptide Y cells, mainly in CA1. Whole cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/- and Nrp2-/- mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2 deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration and higher severity in Nrp2-/- compared to Nrp2+/+ animals. Finally, Nrp2+/- and Nrp2-/-, but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2-/- mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

neuroscience↗

bioRxiv2021

Differential Activity-Dependent Scaling of Synaptic Inhibition and Parvalbumin Interneuron Recruitment in Dentate Projection Neuron Subtypes

Strong inhibitory synaptic gating of dentate gyrus granule cells (GCs), attributed largely to fast-spiking parvalbumin interneurons (PV-INs), is essential to maintain sparse network activity needed for dentate dependent behaviors. However, the contribution of PV-INs to basal and input driven sustained synaptic inhibition in GCs and semilunar granule cells (SGCs), a sparse morphologically distinct dentate projection neuron subtype are currently unknown. In studies conducted in hippocampal slices from mice, we find that although basal inhibitory postsynaptic currents (IPSCs) are more frequent in SGCs and optical activation of PV-INs elicited IPSCs in both GCs and SGCs, optical suppression of PV-INs failed to reduce IPSC frequency in either cell type. Amplitude and kinetics of IPSCs evoked by perforant path activation were not different between GCs and SGCs. However, the robust increase in sustained polysynaptic IPSCs elicited by paired afferent stimulation was lower in SGCs than in simultaneously recorded GCs. Optical suppression of PV-IN selectively reduced sustained IPSCs in SGCs but not in GCs. These results demonstrate that PV-INs, while contributing minimally to basal synaptic inhibition in both GCs and SGCs in slices, mediate sustained feedback inhibition selectively in SGCs. The temporally selective blunting of activity-driven sustained inhibitory gating of SGCs could support their preferential and persistent recruitment during behavioral tasks. Significance StatementOur study identifies that feedback inhibitory regulation of dentate semilunar granule cells, a sparse and functionally distinct class of projection neurons, differs from that of the classical projection neurons, granule cells. Notably, we demonstrate relatively lower activity dependent increase in sustained feedback inhibitory synaptic inputs to semilunar granule cells when compared to granule cells which would facilitate their persistent activity and preferential recruitment as part of memory ensembles. Since dentate granule cell activity levels during memory processing are heavily shaped by basal and feedback inhibition, the fundamental differences in basal and evoked sustained inhibition between semilunar granule cells and granule cells characterized here provide a framework to reorganize current understanding of the dentate circuit processing.

neuroscience↗

bioRxiv2020

Rational Design and Synthesis of Selective PRMT4 Inhibitors: a New Chemotype for Development of Cancer Therapeutics

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates arginine residues of histone H3 and non-histone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and potentially therapeutics. While several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Here, we report the structure-based design of a new class of alanine containing 3-arylindoles as potent and selective PRMT4 inhibitors and describe key structure activity relationships for this class of compounds.

pharmacology and toxicology↗

bioRxiv2020

ExBoX: a simple Boolean exclusion strategy to drive expression in neurons

The advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here we build on previous knowledge to develop a simple AAV-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This Expression by Boolean Exclusion (ExBoX) system encodes for a gene of interest which is turned On by a particular recombinase (Cre or FlpO) and turned Off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally-delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems. Summary statementUbina et al. describe the generation of a novel AAV-based intersectional approach to define and target specific subpopulations of cells in time and space via Expression by Boolean Exclusion (ExBoX).

neuroscience↗

bioRxiv2019

Semilunar Granule Cells Maintain Distinct Dendritic Morphology and Inhibition through Postnatal Development and Receive Heightened Inhibition in Adolescence

Semilunar granule cells (SGCs) have been proposed as a morpho-functionally distinct class of hippocampal dentate projection neurons contributing to feedback inhibition and memory processing in juvenile rats. However, the structural and physiological features that can reliably classify granule cells (GCs) from SGCs through postnatal development remain unresolved. Focusing on postnatal days 11-13, 28-42, and >120, corresponding with human infancy, adolescence, and adulthood, we examined the somatodendritic morphology and inhibitory regulation in SGCs and GCs to determine the cell-type specific features. Unsupervised cluster analysis confirmed that morphological features reliably distinguish SGCs from GCs irrespective of animal age. SGCs maintain higher spontaneous inhibitory postsynaptic current (sIPSC) frequency than GCs from infancy through adulthood. Although sIPSC frequency in SGCs was particularly enhanced during adolescence, sIPSC amplitude and cumulative charge transfer declined from infancy to adulthood and were not different between GCs and SGCs. Extrasynaptic GABA current amplitude peaked in adolescence in both cell types and was significantly greater in SGCs than in GCs only during adolescence. Although GC input resistance was higher than in SGCs during infancy and adolescence, input resistance decreased with developmental age in GCs while it progressively increased in SGCs. Consequently, GCs input resistance was significantly lower than SGCs in adults. The data delineate the structural features that can reliably distinguish GCs from SGCs through development. The results reveal developmental differences in passive membrane properties and steady state inhibition between GCs and SGCs which could confound their use in classifying the cell types.

neuroscience↗

bioRxiv2019

Distinct cellular mediators drive the Janus Faces of Toll-like Receptor 4 regulation of network excitability which impacts working memory performance after brain Injury

The mechanisms by which the neurophysiological and inflammatory responses to brain injury contribute to memory impairments are not fully understood. Recently, we reported that the innate immune receptor, toll-like receptor 4 (TLR4) enhances AMPA receptor (AMPAR) currents and excitability in the dentate gyrus after fluid percussion brain injury (FPI) while limiting excitability in controls. Here we examine the cellular mediators underlying TLR4 regulation of dentate excitability and its impact on memory performance. In ex vivo slices, astrocytic and microglial metabolic inhibitors selectively abolished TLR4 antagonist modulation of excitability in controls, without impacting FPI rats, demonstrating that glial signaling contributes to TLR4 regulation of excitability in controls. In glia-depleted neuronal cultures from naive mice, TLR4 ligands bidirectionally modulated AMPAR charge transfer demonstrating the ability of neuronal TLR4 to regulate excitability, as observed after brain injury. In vivo TLR4 antagonism reduced early post-injury increases in mediators of MyD88-dependent and independent TLR4 signaling without altering expression in controls. Blocking TNF, a downstream effector of TLT4, mimicked effects of TLR4 antagonist and occluded TLR4 agonist modulation of excitability in slices from both control and FPI rats. Functionally, transiently blocking TLR4 in vivo improved impairments in working memory observed one week and one month after FPI, while the same treatment impaired memory function in uninjured controls. Together these data identify that distinct cellular signaling mechanisms converge on TNF to mediate TLR4 modulation of network excitability in the uninjured and injured brain and demonstrate a role for TLR4 in regulation of working memory function. HighlightsO_LITLR4 suppresses dentate excitability in controls through signaling involving glia C_LIO_LINeuronal TLR4 signaling underlies enhanced dentate excitability after brain injury C_LIO_LITNF contributes to TLR4 regulation of excitability in the injured brain C_LIO_LIAltering TLR4 signaling impacts working memory performance C_LIO_LITLR4 signaling is a potential target to improve working memory after brain trauma C_LI

neuroscience↗

bioRxiv2019

Neuronal TLR4 signaling enhances AMPA currents and drives posttraumatic epileptogenesis

Traumatic brain injury is a major risk factor for acquired epilepsies and understanding the mechanisms underlying the early pathophysiology could yield viable therapeutic targets. Growing evidence indicates a role for inflammatory signaling in modifying neuronal excitability and promoting epileptogenesis. Here, we identify that signaling through an innate immune receptor, toll-like receptor 4 (TLR4), in neurons, augments calcium-permeable AMPA receptor (CP-AMPAR) currents in the hippocampal dentate gyrus after brain injury. Blocking TLR4 signaling in vivo shortly after brain injury reduced dentate network excitability and seizure susceptibility. When blocking of TLR4 signaling after injury was delayed, however, this treatment failed to reduce post-injury seizure susceptibility. Further, TLR4 signal blocking was less efficacious in limiting seizure susceptibility when AMPAR currents, downstream targets of TLR4 signaling, were transiently enhanced. Paradoxically, blocking TLR4 signaling augmented both network excitability and seizure susceptibility in uninjured controls. Despite the differential effect on seizure susceptibility, TLR4 antagonism suppressed cellular inflammatory responses after injury without impacting sham controls. These findings demonstrate that independently of glia, the immune receptor TLR4 directly regulates post-traumatic neuronal excitability. Moreover, the TLR4-dependent early increase in dentate excitability is causally associated with epileptogenesis. Identification and selective targeting of the mechanisms underlying the aberrant TLR4-mediated increase in CP-AMPAR signaling after injury may prevent epileptogenesis after traumatic brain injury. Graphical AbstractO_ST_ABSSummary of interactions between TLR4 signaling and brain injury on network excitability and epileptogenesisC_ST_ABSGraphic illustration of the effect of injury and early TLR4 antagonist treatment on early network excitability and the long-term network state. The schematic neurons include TLR4 and AMPAR subunit expression profiles in the acute phase of sham or brain injury. The corresponding early effects on network excitability are depicted by schematic population response traces (inset on upper left). Note the increase in excitability of the uninjured neuron after TLR4 antagonism without changes in AMPAR expression. Note also the increase in TLR4, calcium permeable AMPARs and population excitability after injury and its reduction by TLR4 antagonist treatment. Ampakine enhancement of excitability during TLR4 antagonism is illustrated. The early phase responses and manipulations (including injury, treatments, and molecular responses) are superimposed on a two-tone color-coded network state topology where green indicates low-normal network excitability, ensuring network stability and low risk for epilepsy (Inset on upper right). Note the correspondence between early excitability state (population response profile) and long-term seizure susceptibility and the effects of pharmacological manipulations.

neuroscience↗

bioRxiv2019

Identification and Characterization of the First Fragment Hits for SETDB1 Tudor Domain

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase, which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the TTD of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.

pharmacology and toxicology↗

bioRxiv2018

Identification and Structure-Activity Relationship of HDAC6 Zinc-finger Ubiquitin Binding Domain Inhibitors

HDAC6 plays a central role in the recruitment of protein aggregates for lysosomal degradation, and is a promising target for combination therapy with proteasome inhibitors in multiple myeloma. Pharmacologically displacing ubiquitin from the zinc-finger ubiquitin-binding domain (ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition. Here, we present the discovery of a HDAC6 ZnF-UBD-focused chemical series and its progression from virtual screening hits to low micromolar inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin, and an extended aromatic system stacking with W1182 and R1155 are necessary for activity. One of the compounds induced a conformational remodeling of the binding site where the primary binding pocket opens-up onto a ligand-able secondary pocket that may be exploited to increase potency. The preliminary structure-activity relationship accompanied by nine crystal structures should enable further optimization into a chemical probe to investigate the merit of targeting the ZnF-UBD of HDAC6 in multiple myeloma and other diseases.

biophysics↗