Biology subjects

Basta, T.

Publications and source records attributed to Basta, T..

5 recordsLinked to original sources

bioRxiv2026

Synaptotagmin isoforms differentially regulate glutamate and GABA release in the lateral habenula

Neurotransmitter co-transmission contributes to diverse physiological processes throughout the mammalian brain, including sensory integration, motivational control, and social behaviors. Projections from the globus pallidus internus (GPi; the entopeduncular nucleus, EPN, in rodents) to the lateral habenula (LHb) are well-characterized by the co-transmission of both GABA and glutamate. These dual-release inputs modulate behavioral states in chronically learned helpless (cLH) rats, influencing both the onset and recovery of pathological phenotypes. Here, we employed confocal 3D reconstructions that confirmed the presence of both vesicular transporters VGAT and VGLUT2 in EPN axon terminals within the LHb. Further investigation revealed that GABA and glutamate are packaged in distinct vesicle populations within individual presynaptic terminals. Notably, the calcium (Ca{superscript 2}) sensors Synaptotagmin-2 (Syt2) and Synaptotagmin-3 (Syt3) are highly expressed in the EPN whereas expression of the canonical Ca{superscript 2} sensor, Synaptotagmin 1 (Syt1), is downregulated. Additionally, using confocal microscopy, we observed selective spatial correlations of Syt2 and VGLUT2 and between Syt3 and VGAT in LHb axon terminals. These observations strongly suggested that Syt2 serves as the predominant Ca{superscript 2} sensor for glutamatergic vesicle fusion, and Syt3 serves as the predominant Ca{superscript 2} sensor for GABAergic vesicle fusion in the LHb. To test this hypothesis, we employed targeted antisense oligonucleotide (ASO) knockdown of Syt2 and Syt3 in EPN neurons and measured LHb glutamatergic and GABAergic currents. Syt2 knockdown resulted in an increase in mEPSC frequency, amplitude, half-width and decay, suggesting increased glutamate vesicle release probability and increased glutamate vesicle packing. However, Syt2 knockdown had no influence on mIPSCs amplitude or frequency. On the other hand, Syt3 knockdown had no apparent effect on glutamate release but caused an increase in mIPSC frequency suggesting increased quantal release probability of GABA. Together, these findings identify a molecular mechanism by which synaptotagmin isoforms govern differential glutamate and GABA release at EPN dual-transmitter terminals in the LHb. These results provide evidence for presynaptic mechanisms regulating excitatory-inhibitory balance within this brain structure and importantly provide molecular targets for pharmacological intervention.

neuroscience↗

bioRxiv2025

Localised activity of reverse gyrase at gene regulatory elements

DNA topoisomerases are essential enzymes found in all cells, where they regulate DNA supercoiling. Reverse gyrase (RG) is a unique type of topoisomerase that introduces positive supercoils into DNA and appears exclusively in hyperthermophiles where it was proposed to play a key, yet still elusive, role. Here, we investigate RG activity in the hyperthermophilic archaeon Thermococcus kodakarensis at 85{degrees}C, its optimal growth temperature, using genetics and functional genomics assays. Deletion of RG led to a loss of positive supercoiling in plasmid DNA and the reduced dynamic range of transcription, without affecting histone occupancy. To investigate the effects of RG loss on the topology of chromosomal DNA, we established a psoralen photobinding assay (TMP-seq) in T. kodakarensis under native growth conditions. TMP enrichment patterns were consistent with the twin-domain model of transcription and further revealed that promoters of expressed transcription units are, on average, underwound. TMP-seq profiles in an RG deletion strain revealed that promoters are hotspots for RG activity, consistent with RG acting on hyper-negatively supercoiled substrates. We propose that RG acts not as a global modulator of supercoiling, but as a local genome guardian, selectively stabilising vulnerable regulatory regions to ensure a delicate balance between DNA accessibility and integrity under extremely high temperatures.

microbiology↗

bioRxiv2025

N-Linked Glycosylation as a Driver of Tau Pathology and Neuronal Transmission

N-Linked glycosylation of Tau is implicated in Alzheimers disease, yet how it influences Tau propagation and neuronal uptake remains unclear. Here, we use chemically defined N-glycoforms of 2N4R Tau and the K18 repeat domain to map how glycan site and structure reprogram Tau-polyanion binding, seeding, and entry into human neurons. Site-specific GlcNAc or high-mannose glycans at Asn359 or Asn410 remodel heparin and RNA binding kinetics in biolayer interferometry assays, revealing avidity-dominated, slow-off heparin interactions that are dampened by N-glycans and faster RNA interactions that can be potentiated by small GlcNAc modifications. Cross-seeding experiments show that N-glycosylation selectively tunes nucleation compatibility between Tau proteoforms; GlcNAc at Asn410 in full-length Tau and small glycans at Asn359 in K18 most strongly accelerate aggregation. In HEK293T FRET biosensor cells and iPSC-derived cortical neurons, N-glycans enhance or suppress seeding and pHrodo-reported uptake in a site- and glycan-dependent manner, with Asn410 glycosylation markedly potentiating cellular seeding. Pharmacological inhibition with atropine, dynasore, and cytochalasin D further indicates that N-glycans rebalance muscarinic, dynamin-dependent, and actin-dependent uptake routes. Together, these data establish N-linked glycosylation as a tunable regulator of Tau trafficking and prion-like spread, coupling extracellular receptor usage to intracellular cofactor engagement, and reveals druggable nodes in uptake.

neuroscience↗

bioRxiv2022

A paralog of Pcc1 is the fifth core subunit of KEOPS complex in Archaea

In Archaea and Eukaryotes, the synthesis of a universal tRNA modification, t6A, is catalyzed by the KEOPS complex composed of Kae1, Bud32, Cgi121 and Pcc1. A fifth subunit, Gon7, is found only in Fungi and Metazoa. Mutations in all five genes encoding human KEOPS subunits leads to Galloway-Mowat syndrome, a severe genetic disease causing childhood lethality. Here, we describe the discovery and biochemical characterization of the archaeal fifth KEOPS subunit. This protein, dubbed Pcc2, is a paralog of Pcc1 and is widely conserved in Archaea. Pcc1 and Pcc2 form a heterodimer in solution, show modest sequence conservation but very high structural similarity. The 5-subunit KEOPS lost its capacity to form dimers but its tRNA binding and t6A synthetic activity remained robust. Pcc2 can substitute Pcc1 but the resulting KEOPS complex is inactive suggesting a distinct function for the two paralogs. Comparative sequence and structure analyses point to a possible evolutionary link between archaeal Pcc2 and eukaryotic Gon7 proteins. Our work thus reveals that Pcc2 has evolved to regulate the oligomeric state of KEOPS complex thus adding another layer of complexity to the biosynthesis of t6A that seems to be conserved from Archaea to Eukaryotes.

biochemistry↗

bioRxiv2017

Synaptophysin is a β-Amyloid Target that Regulates Synaptic Plasticity and Seizure Susceptibility in an Alzhiemer’s Model

Intro/abstractAlzheimers disease (AD), a condition characterized by cognitive deficits and progressive loss of memory, is causally linked to the short amyloid peptide A{beta}42, which disrupts normal neurotransmission1,2. Neurotransmitter (NT) release from synaptic vesicles (SV) requires coordinated binding of the conserved core secretory machinery comprised of the soluble NSF attachment protein receptor (vSNARE) synaptobrevin 2 (VAMP2) on the SV and the cognate tSNAREs on the plasma membrane. Synaptophysin (SYP) is the most abundant SV protein3 and the major pre-fusion binding partner of VAMP24. A major challenge in understanding the etiology and prevention of AD is determining the proteins directly targeted by A{beta}42 and elucidating if these targets mediate disease phenotypes. Here we demonstrate that A{beta}42 binds to SYP with picomolar affinity and disrupts the SYP/VAMP2 complex resulting in inhibition of both neurotransmitter release and synaptic plasticity. While functionally redundant paralogs of SYP have masked its critical activity in knockout studies5,6, we now demonstrate a profound seizure susceptibility phenotype in SYP knockout mice that is recapitulated in an AD model mouse. Our studies imply a subtle yet critical role for SYP in the synaptic vesicle cycle and the etiology of AD.

neuroscience↗