Is DNA repair controlled by a biological logic circuit?

The possible utilization of biological logic circuit(s) in the integration and regulation of DNA repair is discussed. The author believes this mode of regulation likely applies to many other areas of cell biology; however, there are currently more experimental data to support its involvement in the control of DNA repair. Sequential logic processes always require a clock to orchestrate the orderly processing of events. In the proposed hypothesis, the pulses in the expression of p53 serve this function. Given the many advantages of logic type control, one would expect that in the course of ~ 3 billion years of evolution, where single cell life forms were likely the only forms of life, a biological logic type control system would have evolved to control at least some biological processes. Several other required components in addition to the ‘clock’ have been identified, such as; a method to temporarily inactivate repair processes when they are not required (e.g. the reversible inactivation of MGMT, a suicide repair protein, by phosphorylation); this prevents complex DNA repair systems with potentially overlapping repair functions from interfering with each other.

Unravelling the regulation pathway of photosynthetic AB-GAPDH

Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light–harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde–3–phosphate dehydrogenase (GAPDH) is a key enzyme of the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant formed by hetero-tetramers of A and B–subunits, and the homotetramer A4. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and for this reason is strictly regulated. While A4–GAPDH is regulated by CP12, AB–GAPDH is autonomously regulated through the C-terminal extension (CTE) of B–subunits. Reversible inactivation of AB–GAPDH occurs via oxidation of a cysteine pair located in the CTE, and substitution of NADP(H) with NAD(H) in the cofactor binding domain. These combined conditions lead to a change in the oligomerization state and enzyme inactivation. SEC–SAXS and single–particle cryoEM analysis disclosed the structural basis of this regulatory mechanism. Both approaches revealed that (A2B2)n–GAPDH oligomers with n=1, 2, 4 and 5 co–exist in a dynamic system. B–subunits mediate the contacts between adjacent A2B2 tetramers in A4B4 and A8B8 oligomers. The CTE of each B–subunit penetrates into the active site of a B–subunit of the adjacent tetramer, while the CTE of this subunit moves in the opposite direction, effectively preventing the binding of the substrate 1,3–bisphosphoglycerate in the B–subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H) by removing NADP(H) from A–subunits allows the entrance of the CTE in B–subunits active sites and hence inactive oligomer stabilization.

Reversible inactivation of ferret auditory cortex impairs spatial and non-spatial hearing

A central question in auditory neuroscience is how far brain regions are functionally specialized for processing specific sound features such as sound location and identity. In auditory cortex, correlations between neural activity and sounds support both the specialization of distinct cortical subfields, and encoding of multiple sound features within individual cortical areas. However, few studies have tested the causal contribution of auditory cortex to hearing in multiple contexts. Here we tested the role of auditory cortex in both spatial and non-spatial hearing. We reversibly inactivated the border between middle and posterior ectosylvian gyrus using cooling (n = 2) or optogenetics (n=1) as ferrets discriminated vowel sounds in clean and noisy conditions. Animals with cooling loops were then retrained to localize noise-bursts from multiple locations and retested with cooling. In both ferrets, cooling impaired sound localization and vowel discrimination in noise, but not discrimination in clean conditions. We also tested the effects of cooling on vowel discrimination in noise when vowel and noise were colocated or spatially separated. Here, cooling exaggerated deficits discriminating vowels with colocalized noise, resulting in increased performance benefits from spatial separation of sounds and thus stronger spatial release from masking during cortical inactivation. Together our results show that auditory cortex contributes to both spatial and non-spatial hearing, consistent with single unit recordings in the same brain region. The deficits we observed did not reflect general impairments in hearing, but rather account for performance in more realistic behaviors that require use of information about both sound location and identity.

Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex [1,2]. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for a diverse group of +TIPs that control microtubule dynamics and interactions with other intracellular structures [3]. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation [4-6] the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, π-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells [7]. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

A chemical-genetic investigation of BDNF-NtrkB signaling in mammalian sleep

Study Objectives The neurotrophin brain derived neurotrophic factor (BDNF) is hypothesized to be a molecular mediator of mammalian sleep homeostasis. This hypothesis is supported by correlational findings and results obtained from pharmacology. BDNF binds with high affinity to the membrane bound receptor Neurotrophin Tyrosine Kinase Receptor B (NtrkB), which triggers several intracellular signaling cascades. It is therefore possible that BDNF’s role in sleep homeostasis is mediated via NtrkB. We examined this hypothesis using a chemical-genetic technique that allows for rapid and selective inhibition of NtrkB in vivo. Methods We used mutant mice bearing a point mutation in the NtrkB that allows for selective and reversible inactivation in the presence of a small binding molecule (1-NM-PP1). Using a cross-over design, we determined the effects of NtrkB inhibition on baseline sleep architecture and sleep homeostasis. Results We find that NtrkB inhibition reduced REM sleep time and increased state-transitions but had no effect on sleep homeostasis. Conclusions These findings suggest that BDNF-NtrkB receptor signaling has relatively subtle roles in sleep architecture, but no role in sleep homeostasis.

Dorsal striatum and the temporal expectancy of an aversive event in Pavlovian odor fear learning

Interval timing, the ability to encode and retrieve the memory of intervals from seconds to minutes, guides fundamental animal behaviors across the phylogenetic tree. In Pavlovian fear conditioning, an initially neutral stimulus (conditioned stimulus, CS) predicts the arrival of an aversive unconditioned stimulus (US, generally a mild foot-shock) at a fixed time interval. Although some studies showed that temporal relations between CS and US events are learned from the outset of conditioning, the question of the memory of time and its underlying neural network in fear conditioning is still poorly understood. The aim of the present study was to investigate the role of the dorsal striatum in timing intervals in odor fear conditioning in male rats. To assess the animal's interval timing ability in this paradigm, we used the respiratory frequency. This enabled us to detect the emergence of temporal patterns related to the odor-shock time interval from the early stage of learning, confirming that rats are able to encode the odor-shock time interval after few training trials. We carried out reversible inactivation of the dorsal striatum before the acquisition session and before a shift in the learned time interval, and measured the effects of this treatment on the temporal pattern of the respiratory rate. In addition, using intracerebral microdialysis, we monitored extracellular dopamine level in the dorsal striatum throughout odor-shock conditioning and in response to a shift of the odor-shock time interval. Contrary to our initial predictions based on the existing literature on interval timing, we found evidence suggesting that transient inactivation of the dorsal striatum may favor a more precocious buildup of the respiratory frequency's temporal pattern during the odor-shock interval in a manner that reflected the duration of the interval. Our data further suggest that the conditioning and the learning of a novel time interval were associated with a decrease in dopamine level in the dorsal striatum, but not in the nucleus accumbens. These findings prompt a reassessment of the role of the striatum and striatal dopamine in interval timing, at least when considering Pavlovian aversive conditioning.

Evolutionary diversification of cytokinin-specific glucosyltransferases in angiosperms and enigma of missing cis-zeatin O-glucosyltransferase gene in Brassicaceae

In the complex process of homeostasis of phytohormones cytokinins (CKs), O-glucosylation catalyzed by specific O-glucosyltransferases represents one of important mechanisms of their reversible inactivation. The CK O-glucosyltransferases belong to a highly divergent and polyphyletic multigene superfamily of glycosyltransferases, of which subfamily 1 containing UDP-glycosyltransferases (UGTs) is the largest in the plant kingdom. It contains recently discovered O and P subfamilies present in higher plant species but not in Arabidopsis thaliana. The cis-zeatin O-glucosyltransferase (cisZOG) genes belong to the O subfamily encoding a stereo-specific O-glucosylation of cis-zeatin-type CKs. We studied different homologous genes, their domains and motifs, and performed a phylogenetic reconstruction to elucidate the plant evolution of the cisZOG gene. We found that the cisZOG homologs do not form a clear separate clade, indicating that diversification of the cisZOG gene took place after the diversification of the main angiosperm families, probably within genera or closely related groups. We confirmed that the gene(s) from group O is(are) not present in A. thaliana and is(are) also missing in the family Brassicaceae. However, cisZOG or its metabolites are found among Brassicaceae clade, indicating that remaining genes from other groups (UGT73—group D and UGT85—group G) are able, at least in part, to substitute the function of group O lost during evolution. This study is the first detailed evolutionary evaluation of relationships among different plant ZOGs within angiosperms.

Cerebellum regulates the timing of periaqueductal grey neural encoding of fear memory and the expression of fear conditioned behaviours

The pivotal role of the periaqueductal grey (PAG) in fear learning is reinforced by the identification of neurons in rat ventral (vPAG) that encode fear memory through signalling the onset and offset of an auditory conditioned stimulus during retrieval. Within this framework, understanding of cerebellar contributions to survival circuits is advanced by the discovery that: (i) reversible inactivation of the medial cerebellar nucleus (MCN) during fear consolidation (a) reduces the temporal precision of vPAG offset, but not onset responses and (b) increases rearing behaviour during retrieval, and (ii) chemogenetic inhibition of the MCN-vPAG projection during fear acquisition (a) reduces the emission of fear-related ultrasonic vocalisations and (b) slows the extinction rate of fear-related freezing. These findings show that the cerebellum regulates fear memory processes at multiple timescales and in multiple ways. The current findings indicate that dysfunctional interactions in the cerebellar-survival network may underlie fear-related disorders and comorbidities.Impact StatementCerebellar-periaqueductal grey interactions contribute to fear conditioned processes and, as such, provide a novel target for treating psychological conditions including post-traumatic stress disorder.

Ventral hippocampus mediates the context-dependence of two-way signaled avoidance in male rats

Considerable work indicates that instrumental responding is context-dependent, but the neural mechanisms underlying this phenomenon are poorly understood. Given the important role for the hippocampal formation in contextual processing, we hypothesized that reversible inactivation of the hippocampus would impair the context-dependence of active avoidance. To test this hypothesis, we used a two-way signaled active avoidance (SAA) task that requires rats to shuttle across a divided chamber during a tone CS in order to avoid a footshock US. After training, avoidance responding was assessed in an extinction test in both the training context and a novel context in a counterbalanced order. Rats performed significantly more avoidance responses in the training context than in the novel context, demonstrating the context-dependence of shuttle avoidance behavior. To examine the role of the hippocampus in the context-dependence of SAA, we reversibly inactivated either the dorsal (DH) or ventral hippocampus (VH) prior to testing. Inactivation of the VH eliminated the context-dependence of SAA and elevated avoidance responding in the novel context to levels similar to that expressed in the training context. In contrast, DH inactivation had no effect on avoidance in either context, and neither manipulation affected freezing behavior. Therefore, the integrity of the VH, but not DH, is required for the expression context-dependence of avoidance behavior.

Dissociable contributions of mediodorsal and anterior thalamic nuclei in visual attentional performance: A comparison using nicotinic and muscarinic cholinergic receptor antagonists

Background: Thalamic subregions mediate various cognitive functions, including attention, inhibitory response control and decision making. Such neuronal activity is modulated by cholinergic thalamic afferents and deterioration of such modulatory signaling has been theorised to contribute to cognitive decline in neurodegenerative disorders. However, the thalamic subnuclei and cholinergic receptors involved in cognitive functioning remain largely unknown. Aims: We investigated whether muscarinic or nicotinic receptors in the mediodorsal thalamus and anterior thalamus contribute to rats’ performance in the five-choice serial reaction time task, which measures sustained visual attention and impulsive action. Methods: Male Long-Evans rats were trained in the five-choice serial reaction time task then surgically implanted with guide cannulae targeting either the mediodorsal thalamus or anterior thalamus. Reversible inactivation of either the mediodorsal thalamus or anterior thalamus were achieved with infusions of the γ-aminobutyric acid-ergic agonists muscimol and baclofen prior to behavioural assessment. To investigate cholinergic mechanisms, we also assessed the behavioural effects of locally administered nicotinic (mecamylamine) and muscarinic (scopolamine) receptor antagonists. Results: Reversible inactivation of the mediodorsal thalamus severely impaired discriminative accuracy and response speed and increased omissions. Inactivation of the anterior thalamus produced less profound effects, with impaired accuracy at the highest dose. In contrast, blocking cholinergic transmission in these regions did not significantly affect five-choice serial reaction time task performance. Conclusions/interpretations: These findings show the mediodorsal thalamus plays a key role in visuospatial attentional performance that is independent of local cholinergic neurotransmission.