Pulsed Feedback Defers Cellular Differentiation

Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable “polyphasic” positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a “timer” that operates over timescales much longer than a cell cycle

PRRT2 controls neuronal excitability by negatively modulating Na+ channel 1.2/1.6 activity

Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c.649dupC mutation. Singlecell patch-clamp experiments on induced pluripotent stem cell-derived neurons from homozygous patients showed increased Na+ currents that were fully rescued by expression of wild-type PRRT2. Closely similar electrophysiological features were observed in primary neurons obtained from the recently characterized PRRT2 knockout mouse. This phenotype was associated with an increased length of the axon initial segment and with markedly augmented spontaneous and evoked firing and bursting activities evaluated, at the network level, by multi-electrode array electrophysiology. Using HEK-293 cells stably expressing Nav channel subtypes, we demonstrated that the expression of PRRT2 decreases the membrane exposure and Na+ current of Nav1.2/Nav1.6, but not Nav1.1, channels. Moreover, PRRT2 directly interacted with Nav1.2/Nav1.6 channels and induced a negative shift in the voltage-dependence of inactivation and a slow-down in the recovery from inactivation. In addition, by co-immunoprecipitation assays, we showed that the PRRT2-Nav interaction also occurs in brain tissue. The study demonstrates that the lack of PRRT2 leads to a hyperactivity of voltage-dependent Na+ channels in homozygous PRRT2 knockout human and mouse neurons and that, in addition to the reported synaptic functions, PRRT2 is an important negative modulator of Nav1.2 and Nav1.6 channels. Given the predominant paroxysmal character of PRRT2-linked diseases, the disturbance in cellular excitability by lack of negative modulation of Na+ channels appears as the key pathogenetic mechanism

Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis

<p>Abstract</p> <p>Background</p> <p>Fluorescent proteins are powerful molecular biology tools that have been used to study the subcellular dynamics of proteins within live cells for well over a decade. Two fluorescent proteins commonly used to enable dual protein labelling are GFP (green) and mCherry (red). Sporulation in the Gram positive bacterium <it>Bacillus subtilis </it>has been studied for many years as a paradigm for understanding the molecular basis for differential gene expression. As sporulation initiates, cells undergo an asymmetric division leading to differential gene expression in the small prespore and large mother cell compartments. Use of two fluorescent protein reporters permits time resolved examination of differential gene expression either in the same compartments or between compartments. Due to the spectral properties of GFP and mCherry, they are considered an ideal combination for co-localisation and co-expression experiments. They can also be used in combination with fluorescent DNA stains such as DAPI to correlate protein localisation patterns with the developmental stage of sporulation which can be linked to well characterised changes in DNA staining patterns.</p> <p>Findings</p> <p>While observing the recruitment of the transcription machinery into the forespore of sporulating <it>Bacillus subtilis</it>, we noticed the occurrence of stage-specific fluorescence intensity differences between GFP and mCherry. During vegetative growth and the initial stages of sporulation, fluorescence from both GFP and mCherry fusions behaved similarly. During stage II-III of sporulation we found that mCherry fluorescence was considerably diminished, whilst GFP signals remained clearly visible. This fluorescence pattern reversed during the final stage of sporulation with strong mCherry and low GFP fluorescence. These trends were observed in reciprocal tagging experiments indicating a direct effect of sporulation on fluorescent protein fluorophores.</p> <p>Conclusions</p> <p>Great care should be taken when interpreting the results of protein localisation and quantitative gene expression patterns using fluorescent proteins in experiments involving intracellular physiological change. We believe changes in the subcellular environment of the sporulating cell leads to conditions that differently alter the spectral properties of GFP and mCherry making an accurate interpretation of expression profiles technically challenging.</p

Russell bodies as a model of ER storage diseases

Protein accumulation represents the consequence of an altered cellular homeostasis leading to an imbalance between synthesis and disposal. Cells adopt a number of strategies to cope with this imbalance: through the Unfolded Protein Response (UPR), intracellular chaperons are increased to facilitate protein folding, and the Endoplasmic Reticulum (ER) expands to accommodate increased concentration of ER resident proteins. The induction of autophagy is also a strategy adopted to face the synthesis of aberrant proteins. Our cellular model of protein accumulation regards mutant immunoglobulin: Ig-\ub5 chain that lacks the first constant domain (\ub5 06CH1 chain). These aberrant chains can neither exit from, nor are efficiently degraded in the ER. As a consequence they accumulate generating dilated cisternae known as Russell bodies. Russell bodies are frequently detected in lymphoproliferative diseases, especially in disorders of secretory B cells. Condensation of aberrant \ub5 06CH1 chains can occur in different sub-cellular locations: when Ig-L chains are produced detergent insoluble aggregates form in the rough ER; without L chains, aggregation occurs in ERGIC compartment. We are interested to define whether and which cellular mechanisms are active or are impaired by the synthesis of aberrant Ig-\ub5 chains assaying: i.e. ER stress, ER expansion, Autophagy modulation in our inducible cellular model (Hela-tet off) of Russell bodies formation. Our aim is to define if Russell bodies structures are a cell defence mechanism against proteotoxicit

PRRT2 modulates presynaptic Ca2+ influx by interacting with P/Q-type channels

Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses

Transient receptor potential vanilloid 1 antagonism in neuroinflammation, neuroprotection and epigenetic regulation: Potential therapeutic implications for severe psychiatric disorders treatment

Transient receptor potential vanilloid 1 (TRPV1) is a polymodal cation channel gated by a large array of chemical and physical stimuli and distributed across different brain regions on neuronal and glial cells. Preclinical studies indicate that TRPV1 might be a target for the treatment of anxiety, depression and addictive disorders. The aim of this narrative review is to focus on studies examining the effects of TRPV1 antagonism on neuroinflammation, neuroprotection and epigenetic regulation. Results suggest that TRPV1 modulation leads to pro- or anti-inflammatory effects depending on the cytokine environment and that the TRPV1 antagonism can switch the microglia towards an anti-inflammatory phenotype. Moreover, TRPV1 inhibitors have neuroprotective properties through the regulation of calcium levels. Finally, TRPV1 antagonism exerts regulatory effects on genes involved in synaptic and cognitive functions through histone deacetylase 2 inhibition. These findings highlight different mechanisms that may underlie the efficacy of TRPV1 antagonists in animal models of severe psychiatric disorders

A novel topology of proline-rich transmembrane protein 2 (PRRT2): Hints for an intracellular function at the synapse

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders

BDNF plasma levels variations in major depressed patients receiving duloxetine

It has been frequently reported that brain-derived neurotrophic factor (BDNF) plays an important role in the pathophysiology of major depressive disorder (MDD). Objective of the study was to investigate BDNF levels variations in MDD patients during antidepressant treatment with duloxetine. 30 MDD patients and 32 healthy controls were assessed using Hamilton Depression Scale (HAM-D) and monitored for BDNF plasma levels at baseline, week 6 and week 12 of duloxetine treatment (60 mg/day) and at baseline, respectively. According to early clinical response to duloxetine (defined at week 6 by reduction >50 % of baseline HAM-D score), MDD patients were distinguished in early responders (ER) and early non-responders (ENR), who reached clinical response at week 12. Laboratory analysis showed significant lower baseline BDNF levels among patients compared to controls. During duloxetine treatment, in ENR BDNF levels increased, reaching values not significantly different compared to controls, while in ER BDNF levels remained nearly unchanged. Lower baseline BDNF levels observed in patients possibly confirm an impairment of the NEI stress-adaptation system and neuroplasticity in depression, while BDNF increase and normalization observed only in ENR might suggest differential neurobiological backgrounds in ER vs. ENR within the depressive syndrom