Early Effects of Alpha-Synuclein Depletion by Pan-Neuronal Inactivation of Encoding Gene on Electroencephalogram Coherence between Different Brain Regions in Mice.

Inactivation of the Snca gene in young mice by chronic injections of tamoxifen (TAM), a selective estrogen receptor modifier, has been shown to decrease the level of alpha-synuclein, a key peptide in the pathogenesis of Parkinson's disease. In young mice, different time courses of the effect were observed in different brain areas, meaning associated disturbances in the intracerebral relations, namely in brain function after TAM-induced synucleinopathy. METHODS: We analyzed electroencephalogram (EEG) coherence ("functional connectivity") between the cortex (MC), putamen (Pt), and dopamine-producing brain regions (ventral tegmental area, VTA, and substantia nigra, SN) in two groups of two-month-old male mice. We compared EEG coherences in the conditional knockout Sncaflox/flox mice with those in their genetic background (C57Bl6J) one, two, and three months after chronic (for five days) intraperitoneal injections of TAM or the vehicle (corn oil). The EEG coherences in the TAM-treated group were compared with those in the alpha-synuclein knockout mice. RESULTS: A significant suppression of EEG coherence in the TAM-treated mice versus the vehicle group was observed in all inter-structural relations, with the exception of MC-VTA at one and three months and VTA-SN at two months after the injections. Suppressive changes in EEG coherence were observed in the alpha-synuclein knockout mice as well; the changes were similar to those in TAM-treated mice three months after treatment. CONCLUSION: our data demonstrate a combined time-dependent suppressive effect induced by TAM on intracerebral EEG coherence.

In FUS[1-359]-tg mice O,S-dibenzoyl thiamine reduces muscle atrophy, decreases glycogen synthase kinase 3 beta, and normalizes the metabolome.

Mutations in the gene encoding the RNA/DNA-binding protein Fused in Sarcoma (FUS) have been detected in familial amyotrophic lateral sclerosis (ALS) patients. FUS has been found to be a critical component of the oxidative damage repair complex that might explain its role in neurodegeneration. Here, we examined what impact antioxidant treatment with thiamine (vitamine B1), or its more bioavailable derivative O,S-dibenzoylthiamine (DBT), would have on the hallmarks of pathology in the FUS[1-359]-transgenic mouse model of ALS. From 8-weeks old, in the pre-symptomatic phase of disease, animals received either thiamine, DBT (200 mg/kg/day), or vehicle for 6 weeks. We examined physiological, behavioral, molecular and histological outcomes, as well as the serum metabolome using nuclear magnetic resonance (NMR). The DBT-treated mice displayed improvements in physiological outcomes, motor function and muscle atrophy compared to vehicle, and the treatment normalized levels of brain glycogen synthase kinase-3ß (GSK-3ß), GSK-3ß mRNA and IL-1ß mRNA in the spinal cord. Analysis of the metabolome revealed an increase in the levels of choline and lactate in the vehicle-treated FUS mutants alone, which is also elevated in the cerebrospinal fluid of ALS patients, and reduced glucose and lipoprotein concentrations in the FUS[1-359]-tg mice, which were not the case in the DBT-treated mutants. The administration of thiamine had little impact on the outcome measures, but it did normalize circulating HDL levels. Thus, our study shows that DBT therapy in FUS mutants is more effective than thiamine and highlights how metabolomics may be used to evaluate therapy in this model.

A bioisostere of Dimebon/Latrepirdine delays the onset and slows the progression of pathology in FUS transgenic mice.

AIMS: To assess effects of DF402, a bioisostere of Dimebon/Latrepirdine, on the disease progression in the transgenic model of amyotrophic lateral sclerosis (ALS) caused by expression of pathogenic truncated form of human FUS protein. METHODS: Mice received DF402 from the age of 42 days and the onset of clinical signs, the disease duration and animal lifespan were monitored for experimental and control animals, and multiple parameters of their gait were assessed throughout the pre-symptomatic stage using CatWalk system followed by a bioinformatic analysis. RNA-seq was used to compare the spinal cord transcriptomes of wild-type, untreated, and DF402-treated FUS transgenic mice. RESULTS: DF402 delays the onset and slows the progression of pathology. We developed a CatWalk analysis protocol that allows detection of gait changes in FUS transgenic mice and the effect of DF402 on their gait already at early pre-symptomatic stage. At this stage, a limited number of genes significantly change expression in transgenic mice and for 60% of these genes, DF402 treatment causes the reversion of the expression pattern. CONCLUSION: DF402 slows down the disease progression in the mouse model of ALS, which is consistent with previously reported neuroprotective properties of Dimebon and its other bioisosteres. These results suggest that these structures can be considered as lead compounds for further optimization to obtain novel medicines that might be used as components of complex ALS therapy.

Familiarity with a vocal category biases the compartmental expression of Arc/Arg3.1 in core auditory cortex.

Learning to recognize a stimulus category requires experience with its many natural variations. However, the mechanisms that allow a category's sensorineural representation to be updated after experiencing new exemplars are not well understood, particularly at the molecular level. Here we investigate how a natural vocal category induces expression in the auditory system of a key synaptic plasticity effector immediate early gene, Arc/Arg3.1, which is required for memory consolidation. We use the ultrasonic communication system between mouse pups and adult females to study whether prior familiarity with pup vocalizations alters how Arc is engaged in the core auditory cortex after playback of novel exemplars from the pup vocal category. A computerized, 3D surface-assisted cellular compartmental analysis, validated against manual cell counts, demonstrates significant changes in the recruitment of neurons expressing Arc in pup-experienced animals (mothers and virgin females "cocaring" for pups) compared with pup-inexperienced animals (pup-naïve virgins), especially when listening to more familiar, natural calls compared to less familiar but similarly recognized tonal model calls. Our data support the hypothesis that the kinetics of Arc induction to refine cortical representations of sensory categories is sensitive to the familiarity of the sensory experience.

Dopamine D4 receptors regulate intracellular calcium concentration in cultured chicken cone photoreceptor cells: relationship to dopamine receptor-mediated inhibition of cAMP formation.

Dopamine is a retinal neuromodulator secreted from amacrine and interplexiform cells. Activation of dopamine D4 receptors on photoreceptor cells reduces a light-sensitive pool of cAMP. The aim of the present study was to evaluate the role of dopamine receptors and cAMP in the regulation of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in photoreceptor cells of chick retina. Retinal cells from 6 day-old chicken embryos were isolated and cultured for 5-7 days prior to experiments. Cone photoreceptors were the predominant cell type in these cultures. Dopamine and agonists of dopamine D4 receptors suppressed K(+)-stimulated uptake of (45)Ca(2+) and [Ca(2+)](i), measured with the Ca(2+)-sensitive fluorescent dye fura-2AM. The effects of the agonists were blocked by dopamine D2/D4 receptor antagonists or by pertussis toxin. 8Br-cAMP, a cell-permeable analog of cAMP, had no effect on inhibition of K(+)-stimulated (45)Ca(2+) influx or [Ca(2+)](i) by dopamine D2/D4 receptor agonists. Quinpirole inhibited the increase in cAMP level elicited by K(+), which requires Ca(2+) influx through voltage-gated Ca(2+) channels, but not that induced by the calcium ionophore A23187. Moreover, dopamine had no effect on either forskolin-stimulated or Ca(2+)/calmodulin-stimulated adenylyl cyclase activity in cell membranes prepared from the cultured cells. These data indicate that the decrease of cAMP elicited by dopamine D4 receptor stimulation may be secondary to decreased [Ca(2+)](i).

Circadian clockwork machinery in neural retina: evidence for the presence of functional clock components in photoreceptor-enriched chick retinal cell cultures.

PURPOSE: Circadian clocks in retinas regulate a variety of biochemical and physiological processes. Retinal neurons, particularly photoreceptor cells, are thought to contain autonomous circadian clocks that control iodopsin expression, cFos expression, cAMP levels, and melatonin synthesis. Photoreceptor-enriched cell cultures prepared from chick embryo retina and entrained to a daily light-dark (LD) cycle exhibit circadian rhythms of cAMP levels and the activity of arylalkylamine N-acetyltransferase (AANAT), a key regulatory enzyme in melatonin synthesis. The present study was conducted to investigate the expression of circadian clockwork machinery comprised of clock genes; a clock-controlled gene, Aanat; and a clock output, melatonin, in the photoreceptor-enriched cultured retinal cells. METHODS: Photoreceptor-enriched cell cultures were prepared from E6 neural retinas and incubated under 14 h:10 h light-dark cycle (LD) of illumination for 8 days and then transferred to constant (24 h/day) darkness (DD). Cells were collected every 4 h in LD and DD, and RNA was isolated. cDNA was prepared from each sample and transcripts of clock genes and Aanat were measured using real-time polymerase chain reaction (PCR). Melatonin release into the culture medium was assayed by HPLC with fluorescence detection at intervals of 3 h in LD and DD. RESULTS: Cultured neural retina cells exposed to a light-dark cycle showed rhythmic expression of clock genes. Bmal1 and Npas2 (also known as Mop4) peaked late in the day in LD and in DD. Clock mRNA was high at night in LD, but arrhythmic in DD. Cry1 and Per2 transcripts increased rapidly in the early morning and were low at night. The rhythm of Per2 was reduced in amplitude in constant darkness (DD). Levels of Cry1 and Per2 transcripts were stimulated by light exposure at night. Melatonin release and Aanat mRNA were low during the day and high at night. Rhythmic expression of clock genes and Aanat was not observed in cultures not exposed to a LD cycle but treated otherwise identically to cultures described above. CONCLUSIONS: Photoreceptor-enriched cell cultures derived from chick embryo neural retina contain a complete circadian clockwork system that is entrained by the light-dark cycle, and has a core timekeeping mechanism and circadian output in the form of melatonin synthesis.

Gating of the cAMP signaling cascade and melatonin synthesis by the circadian clock in mammalian retina.

Melatonin is synthesized in retinal photoreceptor cells and acts as a neuromodulator imparting photoperiodic information to the retina. The synthesis of melatonin is controlled by an ocular circadian clock and by light in a finely tuned mechanism that ensures that melatonin is synthesized and acts only at night in darkness. Here we report that the circadian clock gates melatonin synthesis in part by regulating the expression of the type 1 adenylyl cyclase (AC1) and the synthesis of cAMP in photoreceptor cells. This gating is effected through E-box-mediated transcriptional activation of the AC1 gene, which undergoes robust daily fluctuations that persist in constant illumination. The circadian control of the cAMP signaling cascade indicates that the clock has a more general and profound impact on retinal functions than previously thought. In addition, rhythmic control of AC1 expression was observed in other parts of the central circadian axis, the suprachiasmatic nucleus and pineal gland, but not in other brain areas examined. Thus, clock control of the cAMP signaling cascade may play a central role in the integration of circadian signals that control physiology and behavior.

Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells.

Arylalkylamine N-acetyltransferase (AANAT) is the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. In chicken retina in vivo, AANAT is expressed in a circadian fashion, primarily in photoreceptor cells. AANAT activity is high at night in darkness, low during the daytime, and suppressed by light exposure at night. In the present study, we investigated the circadian and photic regulation of adenosine 3',5'-monophosphate (cAMP) in cultured retinal cells entrained to a daily light-dark (LD) cycle, as well as the role of Ca(2+) and cAMP in the regulation of AANAT activity. Similar to AANAT activity, cAMP levels fluctuate in a daily fashion, with high levels at night in darkness and low levels during the day in light. This daily fluctuation continued with reduced amplitude in constant (24 h/day) darkness (DD). These changes in cAMP appear to be causally related to control of AANAT activity. Adenylyl cyclase and protein kinase A inhibitors suppress the nocturnal increase of AANAT in DD, while 8Br-cAMP augments it. The nocturnal increase of AANAT activity also involves Ca(2+) influx, as it is inhibited by nitrendipine, an inhibitor of L-type voltage-gated channels, and augmented by Bay K 8644, a Ca(2+) channel agonist. The effect of Bay K 8644 was antagonized by the adenylyl cyclase inhibitor MDL 12330A, suggesting a link between Ca(2+) influx, cAMP formation, and AANAT activity in retinal cells. Light exposure at night, which rapidly suppresses AANAT activity, also suppressed cAMP levels. The effect of light on AANAT activity was reversed by Bay K 8644, 8Br-cAMP, and the proteasome inhibitor lactacystin. These results indicate a dynamic interplay of circadian oscillators and light in the regulation of cAMP levels and AANAT activity in photoreceptor cells.

Identification and light-dependent translocation of a cone-specific antigen, cone arrestin, recognized by monoclonal antibody 7G6.

PURPOSE: To elucidate the antigen recognized by monoclonal antibody (mAb) 7G6, a widely used cone-specific marker. METHODS: 7G6 immunocytochemistry was performed on sections of human, primate, and bovine retina. The antigen was immunoprecipitated from human retinal lysates and purified with protein G. Edman degradation and liquid chromatography of tryptic peptides combined with tandem mass spectrometry (LC-MS/MS) identified the antigen. RESULTS: Sequencing of peptides derived from the immunoprecipitated 7G6 antigen identified it as cone arrestin. The identity was confirmed by Western blot analysis with recombinant human cone arrestin and competition with the antibody in immunocytochemistry. Subcellular localization of cone arrestin in dark-adapted and bleached bovine retinas showed that cone arrestin accumulated in cone outer segments of light-adapted retina but was more concentrated in the inner segments of dark-adapted retina. By expression of truncated human cone arrestin mutants systematically deleting areas divergent from bovine and primate cone arrestins, the epitope of 7G6 was identified as a divergent loop exposed at the surface within the N-domain of cone arrestin. CONCLUSIONS: Several independent methods established that the 7G6 antigen is cone arrestin. The 7G6 epitope is contained in a divergent loop, the sequence of which is conserved in bovine and primates but not other vertebrate species consistent with the specificity of the antibody. The light-dependent translocation of cone arrestin suggests a role in light-dark adaptation of cones. Because of the location of its gene on the X-chromosome, cone arrestin is a candidate gene for X-linked cone dystrophies.

Melatonin synthesis in retina: circadian regulation of arylalkylamine N-acetyltransferase activity in cultured photoreceptor cells of embryonic chicken retina.

The key regulatory enzyme in melatonin synthesis is arylalkylamine N-acetyltransferase (AANAT). In vivo, AANAT activity in chicken retinal photoreceptor cells exhibits a circadian rhythm that peaks at night. The purpose of the present study was to investigate the temporal development of light/dark and circadian oscillations of AANAT activity in cultured retinal cells prepared from 6- and 8-day-old chicken embryos (E6, E8, respectively). Photoreceptor cells prepared from E6 retinas and incubated under a 14-h light/10-h dark (LD) cycle of illumination for 5-7 days displayed prominent daily fluctuations in AANAT activity on days 5 and 6 in vitro. However, when E6 cells, incubated for 5 days under LD, were transferred to continuous (24 h/day) darkness (DD) on day 6, no daily pattern of activity was observed. This result indicates that AANAT fluctuations were light-driven and not circadian at this stage. In contrast, cells prepared from E8 embryos and incubated under conditions identical to those for E6 cells displayed prominent rhythms of AANAT activity in both LD and DD, indicative of circadian control. To determine if circadian control of AANAT activity would develop in E6 cells incubated for a longer period of time to allow maturation, cells were incubated for 8 days in LD followed by 2 days in DD. AANAT activity in these cells was rhythmic in both LD and DD. In cells incubated in this manner, a 2-h light pulse in the middle of the subjective night suppressed AANAT activity, indicating that the enzyme activity in the cultured cells is acutely suppressed by light, as it is in vivo. These results indicate that the ability to express circadian regulation of AANAT activity is an intrinsic property of retinal cells that can develop in vitro. Development of light-dark regulation of AANAT activity appears to precede the circadian clock-control of enzyme activity.