Loss of C2orf69 defines a fatal autoinflammatory syndrome in humans and zebrafish that evokes a glycogen-storage-associated mitochondriopathy.

Human C2orf69 is an evolutionarily conserved gene whose function is unknown. Here, we report eight unrelated families from which 20 children presented with a fatal syndrome consisting of severe autoinflammation and progredient leukoencephalopathy with recurrent seizures; 12 of these subjects, whose DNA was available, segregated homozygous loss-of-function C2orf69 variants. C2ORF69 bears homology to esterase enzymes, and orthologs can be found in most eukaryotic genomes, including that of unicellular phytoplankton. We found that endogenous C2ORF69 (1) is loosely bound to mitochondria, (2) affects mitochondrial membrane potential and oxidative respiration in cultured neurons, and (3) controls the levels of the glycogen branching enzyme 1 (GBE1) consistent with a glycogen-storage-associated mitochondriopathy. We show that CRISPR-Cas9-mediated inactivation of zebrafish C2orf69 results in lethality by 8 months of age due to spontaneous epileptic seizures, which is preceded by persistent brain inflammation. Collectively, our results delineate an autoinflammatory Mendelian disorder of C2orf69 deficiency that disrupts the development/homeostasis of the immune and central nervous systems.

Upregulating Lin28a Promotes Axon Regeneration in Adult Mice with Optic Nerve and Spinal Cord Injury.

Severed CNS axons fail to regenerate in adult mammals and there are no effective regenerative strategies to treat patients with CNS injuries. Several genes, including phosphatase and tensin homolog (PTEN) and Krüppel-like factors, regulate intrinsic growth capacity of mature neurons. The Lin28 gene is essential for cell development and pluripotency in worms and mammals. In this study, we evaluated the role of Lin28a in regulating regenerative capacity of diverse populations of CNS neurons in adult mammals. Using a neuron-specific Thy1 promoter, we generated transgenic mice that overexpress Lin28a protein in multiple populations of projection neurons, including corticospinal tracts and retinal ganglion cells. We demonstrate that upregulation of Lin28a in transgenic mice induces significant long distance regeneration of both corticospinal axons and the optic nerve in adult mice. Importantly, overexpression of Lin28a by post-injury treatment with adeno-associated virus type 2 (AAV2) vector stimulates dramatic regeneration of descending spinal tracts and optic nerve axons after lesions. Upregulation of Lin28a also enhances activity of the Akt signaling pathway in mature CNS neurons. Therefore, Lin28a is critical for regulating growth capacity of multiple CNS neurons and may become an important molecular target for treating CNS injuries.

Habenular kisspeptin modulates fear in the zebrafish.

Kisspeptin, a neuropeptide encoded by the KISS1/Kiss1, and its cognate G protein-coupled receptor, GPR54 (kisspeptin receptor, Kiss-R), are critical for the control of reproduction in vertebrates. We have previously identified two kisspeptin genes (kiss1 and kiss2) in the zebrafish, of which kiss1 neurons are located in the habenula, which project to the median raphe. kiss2 neurons are located in the hypothalamic nucleus and send axonal projections to gonadotropin-releasing hormone neurons and regulate reproductive functions. However, the physiological significance of the Kiss1 expressed in the habenula remains unknown. Here we demonstrate the role of habenular Kiss1 in alarm substance (AS)-induced fear response in the zebrafish. We found that AS-evoked fear experience significantly reduces kiss1 and serotonin-related genes (plasmacytoma expressed transcript 1 and solute carrier family 6, member 4) in the zebrafish. Furthermore, Kiss1 administration suppressed the AS-evoked fear response. To further evaluate the role of Kiss1 in fear response, zebrafish Kiss1 peptide was conjugated to saporin (SAP) to selectively inactivate Kiss-R1-expressing neurons. The Kiss1-SAP injection significantly reduced Kiss1 immunoreactivity and c-fos mRNA in the habenula and the raphe compared with control. Furthermore, 3 d after Kiss1-SAP injection, the fish had a significantly reduced AS-evoked fear response. These findings provide an insight into the role of the habenular kisspeptin system in inhibiting fear.

Neuronal connectivity between habenular glutamate-kisspeptin1 co-expressing neurons and the raphe 5-HT system.

The habenula, located on the dorsal thalamic surface, is an emotional and reward processing center. As in the mammalian brain, the zebrafish habenula is divided into dorsal (dHb) and ventral (vHb) subdivisions that project to the interpeduncular nucleus and median raphe (MR) respectively. Previously, we have shown that kisspeptin 1 (Kiss1) expressing in the vHb, regulates the serotonin (5-HT) system in the MR. However, the connectivity between the Kiss1 neurons and the 5-HT system remains unknown. To resolve this issue, we generated a specific antibody against zebrafish Kiss1 receptor (Kiss-R1); using this primary antibody we found intense immunohistochemical labeling in the ventro-anterior corner of the MR (vaMR) but not in 5-HT neurons, suggesting the potential involvement of interneurons in 5-HT modulation by Kiss1. Double-fluorescence labeling showed that the majority of habenular Kiss1 neurons are glutamatergic. In the MR region, Kiss1 fibers were mainly seen in close association with glutamatergic neurons and only scarcely within GABAergic and 5-HT neurons. Our findings indicate that the habenular Kiss1 neurons potentially modulate the 5-HT system primarily through glutamatergic neurotransmission via as yet uncharacterized interneurons. The neuropeptide kisspeptin (Kiss1) play a key role in vertebrate reproduction. We have previously shown modulatory role of habenular Kiss1 in the raphe serotonin (5-HT) systems. This study proposed that the habenular Kiss1 neurons modulate the 5-HT system primarily through glutamatergic neurotransmission, which provides an important insight for understanding of the modulation of 5-HT system by the habenula-raphe pathway.

Kisspeptin1 modulates odorant-evoked fear response via two serotonin receptor subtypes (5-HT1A and 5-HT2) in zebrafish.

Kiss1, a neuropeptide predominantly expressed in the habenula, modulates the serotonin (5-HT) system to decrease odorant cue [alarm substance (AS)]-evoked fear behaviour in the zebrafish. The purpose of this study was to assess the interaction of Kiss1 with the 5-HT system as well as to determine the involvement of the 5-HT receptor subtypes in AS-evoked fear. We utilized 0. 28 mg/kg WAY 100635 (WAY), a selective 5-HT1A receptor antagonist, to observe the effects of Kiss1 administration on AS-evoked fear. We found WAY significantly inhibited the anxiolytic effects of Kiss1 (p < 0.001) with an exception of freezing behaviour. Based on this, we utilized 92.79 mg/kg methysergide, a 5-HT1 and 5-HT2 receptor antagonist, and found that methysergide significantly blocked the anxiolytic effects of Kiss1 in the presence of the AS (p < 0.001). From this, we conclude that Kiss1 modulates AS-evoked fear responses mediated by the 5-HT1A and 5-HT2 receptors. Kiss1 peptide intracranially (IC) administrated has been shown to decrease olfactory, alarm substance (AS)-evoked fear response. Blockade of the 5-HT1A receptor utilizing WAY 100635 (0.28 mg/kg) and the 5-HT1 and 5-HT2 receptor utilizing methysergide (92.79 mg/kg) produced increased AS-evoked fear responses that were unable to be overcome even during the recovery period. Blockade of this 5-HT system followed by Kiss1 administration showed that the peptide was unable to recover the anxiolytic effects upon 5-HT1A blocking using WAY 100635 with the exception of freezing behaviour while methysergide significantly blocked all the anxiolytic effects of Kiss1. These findings implicate that Kiss1 could modulate AS-evoked fear responses mediated by 5-HT1A and 5-HT2 receptors.

Oxidative stress and antioxidant status in primary bone and soft tissue sarcoma.

BACKGROUND: Oxidative stress is characterised by an increased level of reactive oxygen species (ROS) that disrupts the intracellular reduction-oxidation (redox) balance and has been implicated in various diseases including cancer. Malignant tumors of connective tissue or sarcomas account for approximately 1% of all cancer diagnoses in adults and around 15% of paediatric malignancies per annum. There exists no information on the alterations of oxidant/antioxidant status of sarcoma patients in literature. This study was aimed to determine the levels of oxidative stress and antioxidant defence in patients with primary bone and soft tissue sarcoma and to investigate if there exists any significant differences in these levels between both the sarcomas. METHODS: The study cohort consisted of 94 subjects; 20 soft tissue sarcoma, 27 primary bone sarcoma and 47 healthy controls. Malondialdehyde (MDA) and protein carbonyls were determined to assess their oxidative stress levels while antioxidant status was evaluated using catalase (CAT), superoxide dismutase (SOD), thiols and trolox equivalent antioxidant capacity (TEAC). RESULTS: Sarcoma patients showed significant increase in plasma and urinary MDA and serum protein carbonyl levels (p < 0.05) while significant decreases were noted in TEAC, thiols, CAT and SOD levels (p < 0.05). No significant difference in oxidative damage was noted between both the sarcomas (p > 0.05). CONCLUSIONS: In conclusion, an increase in oxidative stress and decrease in antioxidant status is observed in both primary bone and soft tissue sarcomas with a similar extent of damage. This study offers the basis for further work on whether the manipulation of redox balance in patients with sarcoma represents a useful approach in the design of future therapies for bone disease.

Environmental cues determine the fate of astrocytes after spinal cord injury.

Reactive astrogliosis occurs after central nervous system (CNS) injuries whereby resident astrocytes form rapid responses along a graded continuum. Following CNS lesions, naïve astrocytes are converted into reactive astrocytes and eventually into scar-forming astrocytes that block axon regeneration and neural repair. It has been known for decades that scarring development and its related extracellular matrix molecules interfere with regeneration of injured axons after CNS injury, but the cellular and molecular mechanisms for controlling astrocytic scar formation and maintenance are not well known. Recent use of various genetic tools has made tremendous progress in better understanding genesis of reactive astrogliosis. Especially, the latest experiments demonstrate environment-dependent plasticity of reactive astrogliosis because reactive astrocytes isolated from injured spinal cord form scarring astrocytes when transplanted into injured spinal cord, but revert in retrograde to naive astrocytes when transplanted into naive spinal cord. The interactions between upregulated type I collagen and its receptor integrin ß1 and the N-cadherin-mediated cell adhesion appear to play major roles for local astrogliosis around the lesion. This review centers on the environment-dependent plasticity of reactive astrogliosis after spinal cord injury and its potential as a therapeutic target.