The Drosophila functional Smad suppressing element fuss, a homologue of the human Skor genes, retains pro-oncogenic properties of the Ski/Sno family

Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-ß signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, we analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer.

Hypoxia-Ischemia During a Neonatal Sensitive Period Alters Purkinje Neuron Dendritic Complexity Differently in Male and Female Rats

Perinatal hypoxia-ischemia (HI) is a major health issue with no effective therapies beyond head cooling. Notably, male infants are at a greater risk for HI and exhibit more extreme deficits than females. Extensive clinical evidence indicates that perinatal HI impacts the developing cerebellum, yet this region has been largely ignored in preclinical models. Using a modified version of the Rice-Vannucci rat model for HI injury at postnatal day 10, we find reductions in dendritic complexity of Purkinje neurons in males one week later. Females exhibited modest but opposite effects, with slight increases in dendritic complexity, based on Sholl analysis. A custom-made NanoString panel for quantifying mRNAs associated with development, inflammation, and sex differences found almost no commonality in the response to HI in males versus females, with males up-regulating genes associated with microglia activity whereas females increased expression of a protective complement protein, but also of enzymes associated with endocannabinoids and prostaglandins. Both sexes exhibited a reduction in the GABA-synthetic enzymes, GAD-65 and GAD-67, after HI, suggesting increased excitotoxicity, but why males suffered more damage to the Purkinje neurons is unknown.

Development and Optimization of A High-Throughput 3D Rat Purkinje Neuron Culture to Study Paraneoplastic Cerebellar Degeneration

Improved understanding of the mechanisms involved in neurodegenerative disease has been hampered by the lack of robust cellular models that faithfully replicate in vivo features. Here, we present a refined protocol for generating age-dependent, well-developed and synaptically active rat Purkinje neurons in a 3D cell network culture which are responsive to a disease inducer. Using our model, we found that the application of autoantibody Yo, a paraneoplastic cerebellar degeneration (PCD) inducer, alters the structure of the dendritic arbour of cultured Purkinje neurons. The numbers of dendrites per branch-order, the branch-order in itself and the dendritic length were reduced by anti-Yo, proving a functional role for anti-Yo in the pathogenesis of PCD. Our new ex-vivo model is flexible and can be used to investigate disease mechanisms that disturb Purkinje neuron function and communication in 3D. Since it is possible to use the approach in a multi-well format, this method also has high-throughput screening potential.

Deficits Associated With Loss of STIM1 in Purkinje Neurons Including Motor Coordination Can Be Rescued by Loss of Septin 7

Septins are cytoskeletal proteins that can assemble to form heteromeric filamentous complexes and regulate a range of membrane-associated cellular functions. SEPT7, a member of the septin family, functions as a negative regulator of the plasma membrane–localized store-operated Ca2+ entry (SOCE) channel, Orai in Drosophila neurons, and in human neural progenitor cells. Knockdown of STIM, a Ca2+ sensor in the endoplasmic reticulum (ER) and an integral component of SOCE, leads to flight deficits in Drosophila that can be rescued by partial loss of SEPT7 in neurons. Here, we tested the effect of reducing and removing SEPT7 in mouse Purkinje neurons (PNs) with the loss of STIM1. Mice with the complete knockout of STIM1 in PNs exhibit several age-dependent changes. These include altered gene expression in PNs, which correlates with increased synapses between climbing fiber (CF) axons and Purkinje neuron (PN) dendrites and a reduced ability to learn a motor coordination task. Removal of either one or two copies of the SEPT7 gene in STIM1KO PNs restored the expression of a subset of genes, including several in the category of neuron projection development. Importantly, the rescue of gene expression in these animals is accompanied by normal CF-PN innervation and an improved ability to learn a motor coordination task in aging mice. Thus, the loss of SEPT7 in PNs further modulates cerebellar circuit function in STIM1KO animals. Our findings are relevant in the context of identifying SEPT7 as a putative therapeutic target for various neurodegenerative diseases caused by reduced intracellular Ca2+ signaling.

A Facile Approach to Bis(isoxazoles), Promising Ligands of the AMPA Receptor

A convenient synthetic approach to novel functionalized bis(isoxazoles), the promising bivalent ligands of the AMPA receptor, was elaborated. It was based on the heterocyclization reactions of readily available electrophilic alkenes with the tetranitromethane-triethylamine complex. The structural diversity of the synthesized compounds was demonstrated. In the electrophysiological experiments using the patch clamp technique on Purkinje neurons, the compound 1,4-phenylenedi(methylene)bis(5-aminoisoxazole-3-carboxylate) was shown to be highly potent positive modulator of the AMPA receptor, potentiating kainate-induced currents up to 70% at 10−11 M.

Encephalopathy caused by Talisia esculenta intoxication in pregnant ewes and their newborn lambs

An outbreak of acute encephalopathy occurred in pregnant ewes and their newborn lambs associated with consumption of Talisia esculenta fruits and bark. Clinical signs in 5 adult pregnant ewes included drooling, bloat, tachypnea, depression, ataxia, body shaking, difficulty in rising, and recumbency. Three neonatal lambs born to some of those ewes had similar clinical signs. No significant gross abnormalities were observed on autopsy. Histologically, neuronal necrosis, axonal and dendritic swelling, and loss of Purkinje neurons were observed in the cerebellum. The observation of similar neurologic clinical signs and lesions in pregnant ewes and their neonatal lambs suggests that the toxic principle of T. esculenta crosses the placenta and reaches the fetus.

Gjd2b-mediated gap junctions promote glutamatergic synapse formation and dendritic elaboration in Purkinje neurons

Gap junctions between neurons serve as electrical synapses, in addition to conducting metabolites and signaling molecules. During development, early-appearing gap junctions are thought to prefigure chemical synapses, which appear much later. We present evidence for this idea at a central, glutamatergic synapse and provide some mechanistic insights. Loss or reduction in the levels of the gap junction protein Gjd2b decreased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in cerebellar Purkinje neurons (PNs) in larval zebrafish. Ultrastructural analysis in the molecular layer showed decreased synapse density. Further, mEPSCs had faster kinetics and larger amplitudes in mutant PNs, consistent with their stunted dendritic arbors. Time-lapse microscopy in wild type and mutant PNs reveals that Gjd2b puncta promote the elongation of branches and that CaMKII may be a critical mediator of this process. These results demonstrate that Gjd2b-mediated gap junctions regulate glutamatergic synapse formation and dendritic elaboration in PNs.