Mutations in splicing factor PRPF3, causing retinal degeneration, form detrimental aggregates in photoreceptor cells.

PRPF3 is an element of the splicing machinery ubiquitously expressed, yet mutations in this gene are associated with a tissue-specific phenotype: autosomal dominant retinitis pigmentosa (RP). Here, we studied the subcellular localization of endogenous- and mutant-transfected PRPF3. We found that (i) subcellular distribution of the endogenous wild-type protein co-localizes with small nuclear ribonucleoproteins, partially with a nucleolar marker and accumulates in speckles labeled by SC35; (ii) in human retinas, PRPF3 does not show a distinctive abundance in photoreceptors, the cells affected in RP and (iii) the RP causing mutant PRPF3, differently from the wild-type protein, forms abnormally big aggregates in transfected photoreceptor cells. Aggregation of T494M mutant PRPF3 inside the nucleus triggers apoptosis only in photoreceptor cells. On the basis of the observation that mutant PRPF3 accumulates in the nucleolus and that transcriptional, translational and proteasome inhibition can induce this phenomenon in non-photoreceptor cells, we hypothesize that mutation affects splicing factor recycling. Noteworthy, accumulation of the mutant protein in big aggregates also affects distribution of some other splicing factors. Our data suggest that the mutant protein has a cell-specific dominant effect in rod photoreceptors while appears not to be harmful to epithelial and fibroblast cells

Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44.

Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicine generating enzyme, which activates sulfatases by modifying a key cysteine residue within their catalytic domains. SUMF1 is mutated in patients affected by multiple sulfatase deficiency, a rare recessive disorder in which all sulfatase activities are impaired. Despite the absence of canonical retention/retrieval signals, SUMF1 is largely retained in the endoplasmic reticulum (ER), where it exerts its enzymatic activity on nascent sulfatases. Part of SUMF1 is secreted and paracrinally taken up by distant cells. Here we show that SUMF1 interacts with protein disulfide isomerase (PDI) and ERp44, two thioredoxin family members residing in the early secretory pathway, and with ERGIC-53, a lectin that shuttles between the ER and the Golgi. Functional assays reveal that these interactions are crucial for controlling SUMF1 traffic and function. PDI couples SUMF1 retention and activation in the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1 export from and retrieval to the ER, respectively. Silencing ERGIC-53 causes proteasomal degradation of SUMF1, while down-regulating ERp44 promotes its secretion. When over-expressed, each of three interactors favors intracellular accumulation. Our results reveal a multistep control of SUMF1 trafficking, with sequential interactions dynamically determining ER localization, activity and secretion

Enaminone Modulators of Extrasynaptic α4β3δ γ-Aminobutyric AcidA Receptors Reverse Electrographic Status Epilepticus in the Rat After Acute Organophosphorus Poisoning

Seizures induced by organophosphorus nerve agent exposure become refractory to treatment with benzodiazepines because these drugs engage synaptic γ-aminobutyric acid-A receptors (GABAARs) that rapidly internalize during status epilepticus (SE). Extrasynaptic GABAARs, such as those containing α4β3δ subunits, are a putative pharmacological target to comprehensively manage nerve agent-induced seizures since they do not internalize during SE and are continuously available for activation. Neurosteroids related to allopregnanolone have been tested as a possible replacement for benzodiazepines because they target both synaptic and extrasynaptic GABAARs receptors. A longer effective treatment window, extended treatment efficacy, and enhanced neuroprotection represent significant advantages of neurosteroids over benzodiazepines. However, neurosteroid use is limited by poor physicochemical properties arising from the intrinsic requirement of the pregnane steroid core structure for efficacy rendering drug formulation problematic. We tested a non-steroidal enaminone GABAAR modulator that interacts with both synaptic and extrasynaptic GABAARs on a binding site distinct from neurosteroids or benzodiazepines for efficacy to control electrographic SE induced by diisopropyl fluorophosphate or soman intoxication in rats. Animals were treated with standard antidotes, and experimental therapeutic treatment was given following 1 h (diisopropyl fluorophosphate model) or 20 min (soman model) after SE onset. We found that the enaminone 2-261 had an extended duration of seizure termination (>10 h) in the diisopropyl fluorophosphate intoxication model in the presence or absence of midazolam (MDZ). 2-261 also moderately potentiated MDZ in the soman-induced seizure model but had limited efficacy as a stand-alone anticonvulsant treatment due to slow onset of action. 2-261 significantly reduced neuronal death in brain areas associated with either diisopropyl fluorophosphate- or soman-induced SE. 2-261 represents an alternate chemical template from neurosteroids for enhancing extrasynaptic α4β3δ GABAAR activity to reverse SE from organophosphorous intoxication

Neuropeptide S-Mediated Facilitation of Synaptic Transmission Enforces Subthreshold Theta Oscillations within the Lateral Amygdala

The neuropeptide S (NPS) receptor system modulates neuronal circuit activity in the amygdala in conjunction with fear, anxiety and the expression and extinction of previously acquired fear memories. Using in vitro brain slice preparations of transgenic GAD67-GFP (Δneo) mice, we investigated the effects of NPS on neural activity in the lateral amygdala as a key region for the formation and extinction of fear memories. We are able to demonstrate that NPS augments excitatory glutamatergic synaptic input onto both projection neurons and interneurons of the lateral amygdala, resulting in enhanced spike activity of both types of cells. These effects were at least in part mediated by presynaptic mechanisms. In turn, inhibition of projection neurons by local interneurons was augmented by NPS, and subthreshold oscillations were strengthened, leading to their shift into the theta frequency range. These data suggest that the multifaceted effects of NPS on amygdaloid circuitry may shape behavior-related network activity patterns in the amygdala and reflect the peptide's potent activity in various forms of affective behavior and emotional memory

Genomic Analysis of wig-1 Pathways

Background: Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain. Methods and Results: Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes

X-linked Angelman-like syndrome caused by Slc9a6 knockout in mice exhibits evidence of endosomal–lysosomal dysfunction

Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding sodium–hydrogen exchanger 6, a protein mainly expressed in early and recycling endosomes are known to cause a complex and slowly progressive degenerative human neurological disease. Three resulting phenotypes have so far been reported: an X-linked Angelman syndrome-like condition, Christianson syndrome and corticobasal degeneration with tau deposition, with each characterized by severe intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing that a sodium–hydrogen exchanger 6 deficiency would most likely disrupt the endosomal–lysosomal system of neurons, we examined Slc9a6 knockout mice with tissue staining and related techniques commonly used to study lysosomal storage disorders. As a result, we found that sodium–hydrogen exchanger 6 depletion leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons in selective brain regions, most notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and dentate gyrus of the hippocampus and some areas of cerebral cortex. In these select neuronal populations, histochemical staining for β-hexosaminidase activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal disease was observed in the cerebellum and was accompanied by a marked and progressive loss of Purkinje cells, particularly in those lacking the expression of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete clinical phenotype attributable to motor hyperactivity and cerebellar dysfunction. Importantly, these findings show that sodium–hydrogen exchanger 6 loss of function in the Slc9a6-targeted mouse model leads to compromise of endosomal–lysosomal function similar to lysosomal disease and to conspicuous neuronal abnormalities in specific brain regions, which in concert could provide a unified explanation for the cellular and clinical phenotypes in humans with SLC9A6 mutations

The Lysosome and Intracellular Signalling.

In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating the cell's response to nutrient status and growth factor/hormone signalling. The cytosolic surface of the limiting membrane of the lysosome is the site of activation of the multiprotein complex mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates numerous cell growth-related substrates, including transcription factor EB (TFEB). Under conditions in which mTORC1 is inhibited including starvation, TFEB becomes dephosphorylated and translocates to the nucleus where it functions as a master regulator of lysosome biogenesis. The signalling role of lysosomes is not limited to this pathway. They act as an intracellular Ca2+ store, which can release Ca2+ into the cytosol for both local effects on membrane fusion and pleiotropic effects within the cell. The relationship and crosstalk between the lysosomal and endoplasmic reticulum (ER) Ca2+ stores play a role in shaping intracellular Ca2+ signalling. Lysosomes also perform other signalling functions, which are discussed. Current views of the lysosomal compartment recognize its dynamic nature. It includes endolysosomes, autolysosome and storage lysosomes that are constantly engaged in fusion/fission events and lysosome regeneration. How signalling is affected by individual lysosomal organelles being at different stages of these processes and/or at different sites within the cell is poorly understood, but is discussed

Functional studies of PRP3 pre-RNA splicing factor mutated in patients affected by retinitis pigmentosa.

Purpose: Many genes mutated in retinitis pigmentosa (RP) are components of the phototransduction cascade. However, in the last 2 years, genes encoding pre–RNA splicing factors were found mutated in three forms of autosomal dominant RP (adRP, RP11, RP13 and RP18). The slicing factors involved in adRP are PRPF31, PRP3, PRPF8. The link between mutant splicing factors and retinal degeneration is completely obscure. Methods: RNA in situ hybridization studies for the analysis of the expression pattern of PRP3 in embryonic and adult eyes. Subcellular localization of the protein using transfection experiment. Subcellular localization of the mutant forms of Prp3 as found in patients. Results: We analyzed expression of the Prp3 gene in the developing and adult murine eye by RNA in situ hybridization. We found that Prp3 transcripts are ubiquitously distributed but higher amounts could be detected in the central nervous system and neural retina. Expression does not seem to be strictly correlated with the photoreceptor cell layer. Using in vitro transfection in HeLa, NIH3T3 and Cos7 cells, Prp3 localizes into the nuclei. Using similar approaches we found the two different mutations (P493S and T494M) have similar intracellular distribution as found for the wild–type protein. We then wanted to study Prp3 protein distribution compared to splicing proteins. Co–localization analysis of transfected cells using antibodies recognizing a protein of the splicing complex suggests that Prp3 only partially co–localizes with the spliceosome. Conclusions: We analyzed Prp3 transcript distribution in vivo and found no direct correlation with photoreceptors, the cells that undergo degeneration in patients with mutations in this gene. Co–localization with splicing factors confirms that the protein is part of the spliceosome. However the protein shows a more complex subcellular distribution suggesting additional roles of this protein. Finally, mutant Prp3 is translocated into the nucleus like the wild type, ruling out the possibility of mis–folding and degradation of the mutant protein

Functional studies of mutations found in PRPF3 pre-MRNA splicing factor causing RP18

Purpose: Genes encoding pre–mRNA splicing factors (PRPF31, PRPF8, PRPF3) were found mutated in three forms of autosomal dominant RP (adRP, RP11, RP13 and RP18). The link between mutant splicing factors and retinal degeneration is completely obscure. So far, only 2 amino acids (P493S and T494M) were found mutated in PRPF3. Analysis of the subcellular localization and interaction with other splicing factors will be fundamental in defining why mutation of PRPF3 causes RP and whether RP18 is due to a dominant effect of the mutant protein or haploinsufficiency. Methods: Analyses of the subcellular localization of the endogenous protein by immunofluorescence using specific polyclonal and monoclonal antibodies in cell lines and in the murine retina were undertaken. Transfection experiments using cell lines to define subcellular localization of the mutant forms of PRPF3. Interaction of wild type and mutant PRPF3 with known partner proteins and snRNA was performed by co–immunoprecipitation experiments. Results: We analyzed expression of the Prpf3 gene in the developing and adult murine eye by RNA in situ hybridization. We found that Prpf3 transcripts are ubiquitously distributed but higher amounts could be detected in the central nervous system and neural retina. Using in vitro transfection in HeLa, NIH3T3, Cos7 cells and primary retinal cultures, PRPF3 localizes in the nuclei. Using similar approaches we found the two different mutations (P493S and T494M) have similar intracellular distribution as found for the wild–type protein. Mutant PRPF3 also maintains its co–localization with spliceosome specific proteins such as SC35. Conclusions: We analyzed Prpf3 transcript distribution in vivo and found no direct correlation with photoreceptors, the cells that undergo degeneration in patients with mutations in this gene. Co–localization with splicing factors confirms that the protein is part of the spliceosome. Finally, mutant PRPF3 has a subcellular distribution similar to the wild type, suggesting that RP18 is caused either by a dominant effect of the mutation or by haploinsufficiency due to lack of function