Illuminating cAMP dynamics at the synapse with multiphoton FLIM-FRET Imaging

The study of signalling pathways within mammalian physiology has long been hindered by the size of the players involved, being far beyond the realms of the conventional light microscope. The advent of advanced fluorescent imaging techniques has revolutionised our capabilities to probe biological processes. The work in this thesis particularly utilised Förster resonance energy transfer (FRET), a fluorescence-based technique that can provide functional readouts of the processes underlying cellular function. Specifically I worked to develop and optimise a fluorescence imaging system for investigating the dynamics and function of cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger. The neuroscientific study of how the brain can learn and recall memories is a rapidly advancing field. The current challenges of tackling dementias, such as Alzheimer’s disease, and preventing memory loss can only be addressed through better understanding of how memories can be stored. It is now believed that neurons retain memories within their synapses, the femtolitre structures that determine the strength of these connections. cAMP has been shown to play a distinctive role in orchestrating the retention of long term memory at the synaptic level. However, its spatial and temporal activation profiles are still not fully understood. To address this, my PhD project combined FRET readouts with cutting edge imaging techniques applied to synapses in neuronal cultures that provide reasonably convenient optical access. By examining the structure of these synapses, along with the measurement of cAMP concentration in different neuronal regions, this project uncovered the highly compartmentalised nature of this signalling molecule, seen to act directly at the sites of strengthening synapses. Through the optimisation of a FRET imaging system for studying activity in neuronal tissues, this project establishes a method for the future investigation of a plethora of pathways underlying the healthy functioning of the mammalian brain.Open Acces

In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging

Indiana University-Purdue University Indianapolis (IUPUI)The sympathetic nervous system strongly modulates the contractile and electrical function of the heart. The anatomical underpinnings that enable a spatially and temporally coordinated dissemination of sympathetic signals within the cardiac tissue are only incompletely characterized. In this work we took the first step of unraveling the in situ 3D microarchitecture of the cardiac sympathetic nervous system. Using a combination of two-photon excitation fluorescence microscopy and computer-assisted image analyses, we reconstructed the sympathetic network in a portion of the left ventricular epicardium from adult transgenic mice expressing a fluorescent reporter protein in all peripheral sympathetic neurons. The reconstruction revealed several organizational principles of the local sympathetic tree that synergize to enable a coordinated and efficient signal transfer to the target tissue. First, synaptic boutons are aligned with high density along much of axon-cell contacts. Second, axon segments are oriented parallel to the main, i.e., longitudinal, axes of their apposed cardiomyocytes, optimizing the frequency of transmitter release sites per axon/per cardiomyocyte. Third, the local network was partitioned into branched and/or looped sub-trees which extended both radially and tangentially through the image volume. Fourth, sub-trees arrange to not much overlap, giving rise to multiple annexed innervation domains of variable complexity and configuration. The sympathetic network in the epicardial border zone of a chronic myocardial infarction was observed to undergo substantive remodeling, which included almost complete loss of fibers at depths >10 µm from the surface, spatially heterogeneous gain of axons, irregularly shaped synaptic boutons, and formation of axonal plexuses composed of nested loops of variable length. In conclusion, we provide, to the best of our knowledge, the first in situ 3D reconstruction of the local cardiac sympathetic network in normal and injured mammalian myocardium. Mapping the sympathetic network connectivity will aid in elucidating its role in sympathetic signal transmisson and processing

Characterization of endogenous Kv1.3 channel isoforms in T cells.

The voltage-gated potassium channel Kv1.3 plays a crucial role in T-cell activation and is considered a promising target for the treatment of autoimmune diseases. However, the lack of reliable antibodies has prevented its accurate detection and study under endogenous conditions, so that most published studies have been conducted in heterologous systems. To address this limitation, we engineered a Jurkat T-cell line expressing endogenous Kv1.3 channels tagged with a signal peptide to investigate the expression and localization of native Kv1.3 channels, and their role associated to T cell physiological responses. Using the CRISPR-Cas9 tool, we inserted a Flag-Myc peptide at the C terminus of the KCNA3 gene. Basal and activated channel expression were assessed through western blot analysis and imaging techniques. Surprisingly, besides the canonical Kv1.3 channel (54 KDa), we identified two additional isoforms with distinct N termini: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms showed upregulation after T-cell activation. Our focus was on characterizing the truncated isoform (short form, SF) as it had not been previously described and could be present in available Kv1.3-/- mouse models. Overexpressing SF in HEK cells generated Kv1.3-like currents with smaller amplitudes, which, unlike canonical Kv1.3, did not induce HEK proliferation. To explore the role of endogenous SF isoform in a native system, we generated both a knockout Jurkat clone and a clone expressing only the SF isoform. While the canonical isoform localized primarily at the plasma membrane, SF remained intracellular, accumulating perinuclearly. Consequently, SF Jurkat cells lacked Kv1.3 currents, exhibited depolarized resting membrane potential (EM), reduced Ca2+ influx, and diminished increases in intracellular calcium ([Ca2+]i) upon stimulation. Functional characterization of these Kv1.3 channel isoforms revealed their differential contributions to signaling pathways involved in immunological synapse formation. In conclusion, alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells with distinct functional roles. Importantly, some of these functions do not require the formation of functional plasma membrane channels by Kv1.3 proteins.El canal de potasio dependiente de voltaje Kv1.3 juega un papel crucial en la activación de las células T y se considera una buena diana terapéutica para el tratamiento de enfermedades autoinmunes. Sin embargo, la falta de anticuerpos específicos de la proteína ha impedido su detección y estudio precisos en condiciones endógenas, por lo que la mayoría de los estudios publicados se han realizado en sistemas heterólogos. Para abordar esta limitación, diseñamos una línea de células T Jurkat que expresa canales Kv1.3 endógenos marcados con un péptido señal para estudiar su expresión y localización además de su papel fisiológico en los linfocitos T. Usando la herramienta CRISPR-Cas9, insertamos un péptido Flag-Myc en el extremo C del gen KCNA3. La expresión del canal basal y activado se evaluó mediante análisis de transferencia Western y técnicas de imagen. Sorprendentemente, además del canal canónico Kv1.3 (54 KDa), identificamos dos isoformas adicionales con extremos N distintos: una isoforma más larga (70 KDa) y una isoforma truncada (43 KDa). Las tres isoformas mostraron un aumento de su expresión después de la activación de las células T. Nuestro objetivo fue caracterizar la isoforma truncada (forma abreviada, SF) ya que no se había descrito previamente y podría estar presente en los modelos de ratón Kv1.3-/- disponibles. La sobreexpresión de SF en células HEK generó corrientes similares a Kv1.3 con amplitudes más pequeñas que, a diferencia del Kv1.3 canónico, no indujeron la proliferación de HEK. Para explorar el papel de la isoforma SF endógena en un sistema nativo, generamos un clon de Jurkat knockout y un clon que expresa solo la isoforma SF. Mientras que la isoforma canónica se localizó principalmente en la membrana plasmática, el SF permaneció intracelular, acumulándose perinuclearmente. En consecuencia, las células SF Jurkat carecían de las corrientes Kv1.3, presentaban un potencial de membrana en reposo (EM) despolarizado, una entrada de Ca2+ reducida y un aumento disminuido del calcio intracelular ([Ca2+]i) tras la estimulación. La caracterización funcional de estas isoformas del canal Kv1.3 reveló sus contribuciones diferenciales a las vías de señalización involucradas en la formación de sinapsis inmunológicas. En conclusión, el inicio de la traducción alternativa genera al menos tres isoformas endógenas del canal Kv1.3 en las células T con distintas funciones funcionales. Es importante destacar que algunas de estas funciones no requieren la formación de canales de membrana plasmática funcionales por las proteínas Kv1.3.Escuela de DoctoradoDoctorado en Investigación Biomédic

Chronic Nicotine Cell Specifically Upregulates Functional α4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose α4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased α4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change α4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional α4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases α4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated α4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional α4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain

CANCER TREATMENT BY TARGETING HDAC4 TRANSLOCATION INDUCED BY MICROSECOND PULSED ELECTRIC FIELD EXPOSURE: MECHANISTIC INSIGHTS THROUGH KINASES AND PHOSPHATASES

Epigenetic modifications, arising from sub-cellular shifts in histone deacetylase (HDAC) activity and localization, present promising strategies for diverse cancer treatments. HDACs, enzymes responsible for post-translational histone modifications, induce these epigenetic changes by removing acetyl groups from ε-N-acetyl-lysine residues on histones, thereby suppressing gene transcription. Within the HDAC group, class IIa HDACs are notable for their responsiveness to extracellular signals, bridging the gap between external stimuli, plasma membrane, and genome through nuclear-cytoplasmic translocation. This localization offers two significant mechanisms for cancer treatment: nuclear accumulation of HDACs represses oncogenic transcription factors, such as myocyte-specific enhancer factor 2C (MEF2C), triggering various cell death pathways. Conversely, cytoplasmic HDAC accumulation acts similarly to HDAC inhibitors by silencing genes. My dissertation introduces an innovative approach for glioblastoma and breast cancer treatment by investigating the application of microsecond pulsed electric fields. It particularly focuses on HDAC4, a class IIa HDAC overexpressed in these cancers. Beyond demonstrating HDAC4 translocation, my research delves into the intricate roles of kinases and phosphatases, shedding light on the underlying factors governing HDAC4 translocation

Dissection of visual signalling based on functionally specific rod photoreceptor mutants

Visual signalling initiates in the rod and cone photoreceptors in the retina. The first steps include a multi-step amplification cascade starting in the outer segments upon light exposure and activating the phosphodiesterase 6 (PDE6). PDE6 reduces the level of the second messenger cyclic guanosine monophosphate (cGMP) by hydrolysis. Low levels of cGMP in turn mediate the closure of cyclic nucleotide-gated (CNG) channels as the final step of phototransduction causing a voltage change, the first electrical signal in rods. At downstream photoreceptor synaptic terminals this leads to a reduction in neurotransmitter release which activates the bipolar cells, retinal second order neurons. Thus, the focus of this thesis was to investigate rod signalling precisely by means of rod-specific mutations in rodent models and electroretinography (ERG) which measures the electrical activity of the retina including photoreceptor outer segment function and signal transmission to bipolar. This work concentrates on the role of four distinctive components located in different compartments of rods from the outer segment to the synaptic terminal. The first part of the thesis addresses the contribution of the PDE6 and the CNG channels, located in rod outer segments, to signal generation. The role of PDE6 was studied in Pde6a mutants with differently compromising missense mutations in the alpha-subunit of rod PDE6 (Pde6a) resulting in a gradually reduced PDE6 activity in each Pde6a variant. This causes a continuously elevated level of cGMP which triggers a premature degeneration of rods and secondary cone cell death. In this regard, we have found that functionally the generation of rod-driven electrical signals are prevented, resulting in a mostly cone-driven vision in Pde6a variants. The remaining cone signal in each Pde6a line is ultimately determined by the speed of photoreceptor degeneration. The role of CNG channels was addressed in the scope of AAV-mediated gene replacement therapy in the Cngb1-/- knockout model characterized by a lack of the beta subunit of the CNG channel (CNGB1) and a respective functional silencing of rod signalling. Functional assessment revealed that genetic restoration of Cngb1 established rod electrical signals which were even translated to second and third order neurons in the retina. The second part concerns the role of the hyperpolarization-activated and cyclic nucleotide-gated channels 1 (HCN1) situated functionally downstream at the inner segments of rod photoreceptors. This work illustrates that a loss of HCN1 channels prolonged rod responses and subsequently saturate rod pathway. Consequently, under regular conditions, HCN1 mediates an inward current which reduces outer segment activity during bright light and enhances rod responsivity. We show here that HCN1 channels are important components of early signal processing within the photoreceptor. The final part of the thesis describes the role of voltage-gated calcium (CaV1.4) channels. These channels control transmitter release at synaptic terminals, the final step of rod signal processing. Our functional studies describe the consequence of two mutations in the CaV1.4 channels on the synaptic activity. A complete loss of the Cacna1f causes a complete failure of signal transmission from photoreceptors to second-order neurons

Identification of novel synaptic components by transcriptome profiling of the murine neuromuscular junction

The neuromuscular junction (NMJ) has been studied for over a century, yet we still do not have a complete picture of all its structural and functional components, knowledge of which is paramount in devising treatment strategies for neuromuscular diseases. Previous microarray-based approaches aimed at elucidating novel NMJ components were hindered by technological limitations. Recent technological advancements propelled next-generation RNA-sequencing with its wider dynamic range to the forefront of transcriptome-level gene expression profiling. We utilized laser-capture microdissection to isolate myonuclei underlying the NMJ combined with RNA-sequencing and successfully generated NMJ gene expression profiles of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles and identified a large number of potential novel NMJ genes. The expression levels of canonical NMJ genes were nearly identical between the EDL and SOL, which suggests that the core NMJ gene program might be well conserved between different skeletal muscle types. We used in vivo muscle electroporation to overexpress one of our candidate genes, the transcription factor T-box 21 (TBX21), in the tibialis anterior (TA) muscle and observed an increased density of postsynaptic acetylcholine receptors. TBX21 may thus represent a novel transcription factor contributing to the regulation of the NMJ gene program, with a role in postsynaptic sensitivity. We also generated NMJ gene expression profiles of the TA muscle of 10-month-old (“young”) and 30-month-old (“old”) mice to investigate the effect of aging on the NMJ gene program. Strikingly, the NMJ gene program was remarkably stable, with nearly identical expression levels of canonical NMJ genes between young and old mice. This implies that age-related perturbations of the NMJ are likely caused by external factors, such as accumulated myofiber damage and changes in nerve input, rather than by gradual dysregulation of the NMJ gene program with increasing age. Our findings argue against the hypothesis that aging leads to a broad deterioration of the NMJ gene program that would contribute to perturbations of NMJ structure and function. Furthermore, functional annotation analysis of our different NMJ gene expression datasets strongly indicates the importance of an extensive number of hitherto unknown glycoproteins, as well as of posttranslational modifications, especially glycosylations, at the synaptic basal lamina. We highlight a set of candidate genes that encode for enzymes putatively involved in these processes at the NMJ, and which are potentially involved in the pathophysiology of neuromuscular diseases such as congenital myasthenic syndromes. This thesis expands our understanding of the complexity of the NMJ and lays the foundation for further research that will functionally characterize novel synaptic components and provide the basis for novel therapeutic treatment strategies

Chronic Nicotine Cell Specifically Upregulates Functional α4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose α4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased α4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change α4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional α4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases α4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated α4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional α4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain

Rapid action of glucocorticoids in mast cells

Glucocorticoids (GCs) rapidly inhibit mast cells function, but the mechanism of action is still poorly understood. A knowledge of how GCs rapidly suppress allergy and inflammation mediated by these cells will aid in the development of new drugs with a better benefit-risk ratio. The aim of this research work was to study the rapid action of GCs in mast cells and investigate the involvement of the glucocorticoid receptor as a mediator of the rapid effects