Biology subjects

Ramirez-Franco, J.

Publications and source records attributed to Ramirez-Franco, J..

6 recordsLinked to original sources

bioRxiv2025

A hyperglycosylated form of Kv1.2 upregulated in LGI1 knockout mice

Kv1 voltage-gated potassium channels determine key functional neuronal properties. Their activity is modulated by subunit composition and post-translational modifications such as phosphorylation and glycosylation. Using an antibody directed against a phosphotyrosine (Y458) located in the C-terminal tail of Kv1.2, we identified yet unreported high molecular weight forms of Kv1.2 among them, a phosphorylated and heavily glycosylated 100 kDa form. Owing to the significant downregulation of Kv1.2 in LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, we investigated, in total brain and the hippocampal formation of both WT and Lgi1-/- mice, the distribution of phosphoY458 Kv1.2 and we compared their respective proteomic interactomes with those of Kv1.2. In addition to major differences between the interactomes of pY458Kv1.2 and Kv1.2 in WT and Lgi1-/-, we found a major reshaping of pY458 Kv1.2 molecular neighbourhood between WT and Lgi1-/- as well as a significant upregulation of the glycosylated form in Lgi1-/-.

neuroscience↗

bioRxiv2023

Molecular Basis of Kv1 Channel Downregulation and Its Epileptogenic Implications in Lgi1 Knock-out Mice

The Kv1 members (KCNA, Shaker) of the voltage-gated potassium channels are implicated in determining key functional neuronal properties from spike generation at axonal initial segments to the control of synaptic strength at nerve terminals. In animal models of LGI1-dependent autosomal dominant lateral temporal lobe epilepsy (ADTLE), Kv1 channels are downregulated, suggesting their crucial involvement in epileptogenesis. The molecular basis of Kv1 channel-downregulation in LGI1 knock-out mice has not been elucidated and how the absence of this extracellular protein induces an important modification in the expression of Kv1 remains unknown. In this study we analyse by immunofluorescence the detailed modifications in neuronal Kv1.1 and Kv1.2 distribution throughout the hippocampal formation of LGI1 knock-out mice. We show that Kv1 downregulation is not restricted to the axonal compartment, but also takes place in the somatodendritic region and is accompanied by a drastic decrease in Kv2 expression levels. Moreover, we find that the downregulation of these Kv channels is associated with an important increase in bursting patterns. Finally, mass spectrometry uncovered key important modifications in the Kv1 interactome that highlight the epileptogenic implication of Kv1 downregulation in LGI1 knock-out animals.

neuroscience↗

bioRxiv2023

Rescue of normal excitability in LGI1-deficient epileptic neurons

Leucine-rich Glioma Inactivated 1 (LGI1) is a glycoprotein secreted by neurons, the deletion of which leads to Autosomal Dominant Lateral Temporal Lobe Epilepsy. Recently, we showed that LGI1 deficiency in a mouse model (KO-Lgi1) decreased Kv1.1 channel density at the axon initial segment (AIS) and at presynaptic terminals, thus enhancing both intrinsic excitability and glutamate release. However, the precise conditions for rescuing normal excitability in KO-Lgi1 neurons have still not been reported. Here we show that the selective expression of LGI1 in KO-Lgi1 neurons with the use of single-cell electroporation reduces intrinsic excitability, and restores both the Kv1.1 mediated D-type current and Kv1.1 immunostaining at the AIS. In addition, we show that the homeostatic shortening of the AIS length observed in KO-Lgi1 neurons is prevented in neurons electroporated with the Lgi1 gene. Furthermore, we reveal a spatial gradient of both intrinsic excitability and Kv1.1 immunostaining that is centred on the electroporated neuron. We conclude that expression of LGI1 restores normal excitability through the expression of functional Kv1 channels at the AIS.

neuroscience↗

bioRxiv2022

Deep learning based image analysis identifies a DAT-negative subpopulation of dopaminergic neurons in the lateral Substantia nigra.

Here we present a deep learning-based image analysis platform (DLAP), tailored to autonomously quantify cell numbers, and fluorescence signals within cellular compartments, derived from RNAscope or immunohistochemistry. We utilized DLAP to analyse subtypes of tyrosine hydroxylase (TH)-positive dopaminergic midbrain neurons in mouse and human brain sections. These neurons modulate complex behaviour, and are differentially affected in Parkinsons and other diseases. DLAP allows the analysis of large cell numbers, and facilitates the identification of small cellular subpopulations. Specifically, we identified a small subpopulation of TH-positive neurons ([~]5%), mainly located in the very lateral Substantia nigra (SN), that was immunofluorescence-negative for the plasmalemma dopamine transporter (DAT), with [~]40% smaller cell bodies. These neurons were negative for aldehyde dehydrogenase 1A1, with a lower co-expression rate for dopamine-D2-autoreceptors, but a [~]7-fold higher likelihood of calbindin-d28k co-expression ([~]70%). Our results have important implications, as DAT is crucial for dopamine-signalling, and is commonly used as a marker for dopaminergic SN neurons.

neuroscience↗

bioRxiv2021

The protein-binding pocket of Botulinum neurotoxin B accommodates a preassembled synaptotagmin / ganglioside complex

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.

pharmacology and toxicology↗

bioRxiv2021

REFINING THE IDENTITY AND ROLE OF KV4 CHANNELS IN MOUSE SUBSTANTIA NIGRA DOPAMINERGIC NEURONS

Substantia nigra pars compacta (SNc) dopaminergic (DA) neurons display a peculiar electrical phenotype characterized in vitro by a spontaneous tonic regular activity (pacemaking activity), a broad action potential and a biphasic post-inhibitory response. Several studies in rodents have underlined the central role played by the transient A-type current (IA) in the control of pacemaking activity and post-inhibitory rebound properties, thereby influencing both DA release and the physiological response of SNc neurons to incoming inhibitory inputs. Kv4.3 potassium channels were considered to be fully responsible for IA in these neurons, their density being tightly related to pacemaking frequency. In spite of this crucial electrophysiological role, we show that Kv4.3-/- transgenic mice exhibit minor alterations in locomotion and motor learning, although no compensation by functionally overlapping ion channels is observed in Kv4.3-/- SNc DA neurons. Using antigen retrieval immunohistochemistry, we further demonstrate that Kv4.2 potassium channels are also expressed in SNc DA neurons, even though their contribution to IA appears significant only in a minority of neurons (~5-10%). Using correlative analysis on recorded electrophysiological parameters and multi-compartment modeling, we then demonstrate that, rather than its conductance level, IA gating kinetics (inactivation time constant) appear as the main biophysical property defining post-inhibitory rebound delay and pacemaking frequency. Moreover, we show that the hyperpolarization-activated current (IH) has an opposing and complementary influence on the same firing features, and that the biophysical properties of IA and IH are likely coregulated in mouse SNc DA neurons. SIGNIFICANCE STATEMENTSubstantia nigra pars compacta (SNc) dopaminergic (DA) neurons are characterized by pacemaking activity, a broad action potential and biphasic post-inhibitory response. The A-type transient potassium current (IA) plays a central role in both pacemaking activity and post-inhibitory response. While it was thought so far that Kv4.3 ion channels were fully responsible for IA, using a Kv4.3-/- transgenic mouse and antigen retrieval immunohistochemistry we demonstrate that Kv4.2 channels are also expressed in SNc DA neurons, although their contribution is significant in a minority of neurons only. Using electrophysiological recordings and computational modeling, we then demonstrate that IA gating kinetics and its functional complementarity with the hyperpolarization-activated current are major determinants of both pacemaking activity and post-inhibitory response in SNc DA neurons.

neuroscience↗