Antipurinergic Therapy Corrects the Autism-Like Features in the Poly(IC) Mouse Model

Background

Autism spectrum disorders (ASDs) are caused by both genetic and environmental factors. Mitochondria act to connect genes and environment by regulating gene-encoded metabolic networks according to changes in the chemistry of the cell and its environment. Mitochondrial ATP and other metabolites are mitokines—signaling molecules made in mitochondria—that undergo regulated release from cells to communicate cellular health and danger to neighboring cells via purinergic signaling. The role of purinergic signaling has not yet been explored in autism spectrum disorders.

Objectives and Methods

We used the maternal immune activation (MIA) mouse model of gestational poly(IC) exposure and treatment with the non-selective purinergic antagonist suramin to test the role of purinergic signaling in C57BL/6J mice.

Results

We found that antipurinergic therapy (APT) corrected 16 multisystem abnormalities that defined the ASD-like phenotype in this model. These included correction of the core social deficits and sensorimotor coordination abnormalities, prevention of cerebellar Purkinje cell loss, correction of the ultrastructural synaptic dysmorphology, and correction of the hypothermia, metabolic, mitochondrial, P2Y2 and P2X7 purinergic receptor expression, and ERK1/2 and CAMKII signal transduction abnormalities.

Conclusions

Hyperpurinergia is a fundamental and treatable feature of the multisystem abnormalities in the poly(IC) mouse model of autism spectrum disorders. Antipurinergic therapy provides a new tool for refining current concepts of pathogenesis in autism and related spectrum disorders, and represents a fresh path forward for new drug development.     

The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

     

Human Multipotent Stromal Cells (MSCs) Increase Neurogenesis and Decrease Atrophy of the Striatum in a Transgenic Mouse Model for Huntington's Disease

Background

Implantation of human multipotent stromal cells from bone marrow (hMSCs) into the dentate gyrus of the hippocampus of mice was previously shown to stimulate proliferation, migration and neural differentiation of endogenous neural stem cells. We hypothesized that hMSCs would be beneficial in a mouse model of Huntington disease (HD) due to these neurogenic effects.

Results

We implanted hMSCs into the striatum of transgenic mice (N171-82Q) that are a model for HD. The implanted hMSCs rapidly disappeared over 3 to 15 days. However, they increased proliferation and neural differentiation of endogenous neural stem cells for up to 30 days. They also increased neurotrophic signaling and decreased atrophy of the striatum in 3-month old HD mice implanted with hMSCs one month earlier.

Conclusions

The results therefore suggested that neural implantation of hMSCs may be of benefit in HD but a number of parameters of dose, treatment schedule, and route of administration need to be optimized.     

Secreted Frizzled-related Protein 1 (Sfrp1) Regulates the Progression of Renal Fibrosis in a Mouse Model of Obstructive Nephropathy

Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1^−/− obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1^−/− mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.     

Genetic and Experiential Features in a Case of Cultural Transmission in the House Mouse (Mus musculus)

The present study analyses the capacity of house mice (Mus musculus) to solve a problem, consisting in opening a door which must be rotated 50 times in the same direction to allow access to the food reward. This capacity emerged spontaneously in 4.8 % of 500 animals belonging to a random bred Swiss population, tested beforehand. The experiment then investigated the effects of genetic and experiential (social) factors on transmission of this capacity from one individual to another. Five groups of approximately 70 animals each were compared: a) controls; b) animals with no experience of the problem but offspring of parents able to solve the problem spontaneously; c) animals with experience of the problem being offspring of parents able to solve it spontaneously; d) naive offspring of parents incapable of solving the problem spontaneously; e) animals with experience of the problem and offspring of parents incapable of solving it. The results showed considerable effects both of genetic and experiential (social) factors with values ranging from only 3.9 % of successful naive mice among offspring of unsuccessful individuals, up to 32.3 % of successful offspring of successful parents, reared with a mother who solved the problem several times in their presence.     

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse,a Model of Leigh Syndrome

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys⁷⁷ and Cys⁴⁸ were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.We previously identified the formation of S-(2-succino)cysteine (2SC)¹ (protein succination) as a result of the irreversible reaction of fumarate with reactive cysteine thiols (1, 2). Fumarate concentrations are increased during adipogenesis and adipocyte maturation (2, 3), and the excess of glucose and insulin leads to augmented protein succination in the adipose tissue of type 2 diabetic mice (4, 5). Protein succination is also specifically increased in fumarate hydratase deficient hereditary leiomyomatosis and renal cell carcinoma (HLRCC), because of the decreased conversion of fumarate to malate (6, 7). In both cases, intracellular fumarate concentrations are elevated; in fumarate hydratase deficient cells, the fumarate concentration is about 5 mm (8), whereas fumarate levels increase up to fivefold in adipocytes grown in the presence of high (30 mm) versus normal (5 mm) glucose concentrations (2). In the adipocyte the increase in fumarate and succinated proteins develops as a direct result of mitochondrial stress induced by nutrient excess. Mechanistically, excess glucose without increased ATP demand inhibits the electron transport chain resulting in an elevated NADH/NAD⁺ ratio. This inhibits NAD⁺-dependent Krebs cycle enzymes and leads to an increase in fumarate and protein succination (9). In support of this we have also shown that low concentrations of chemical uncouplers of oxidative phosphorylation (OXPHOS) can decrease fumarate concentrations and protein succination (9). The physiological consequences of protein succination include a decrease in the functionality of the target protein (8, 10–12), for example succination of adiponectin prevents the formation of multimeric complexes and reduces plasma adiponectin levels in diabetes (4). Considering the impact of glucotoxicity driven mitochondrial stress in the adipocyte, we predicted that deficiencies in OXPHOS associated with NADH accumulation would also result in increased protein succination.Mitochondrial respiratory chain disorders encompass a broad range of encephalopathies and myopathies associated with the defective assembly, activity or maintenance of the OXPHOS machinery (13), and are estimated to occur in about 1 in 5,000 live births (14). A common feature in most mitochondrial diseases (MD) is a failure to thrive because of reduced mitochondrial energy production; both the brain and muscle are usually affected because of their high dependence on oxidative metabolism (13). Leigh syndrome is one of the most common manifestations of MD and is characterized by progressive neurodegeneration with bilateral necrotizing lesions of the brainstem and basal ganglia, resulting in lactic acidosis, ataxia, seizures, dystonia, and respiratory failure (15, 16). Mutations in genes encoding the five complexes of the OXPHOS machinery can lead to Leigh syndrome; however, the majority of these mutations affect subunits of complexes I and IV (17), and both mitochondrial and nuclear encoded proteins may be affected (17–19). Complex I is a large (980 kDa) l-shaped protein assembly consisting of 45 peptides, with one flavin mononucleotide and eight iron–sulfur clusters (20). One of the first identified mutations of complex I encoded Ndufs4, a small (18 kDa) assembly protein (21–23). Ndufs4 assists in the final stages of complex I assembly, and its absence results in the formation of a smaller ∼830 kDa subcomplex that lacks the NADH dehydrogenase module and has significantly less electron shuttling activity than the intact holoenzyme (24, 25). Ndufs4 mutations are associated with brainstem deterioration in humans (26), and a recently described Ndufs4 knockout mouse (Ndufs4 KO) exhibits many of the clinical and neurological symptoms observed in human Leigh syndrome (27, 28).One of the most common clinical features of MD is lactic acidosis, derived from the accumulation of pyruvate and elevated NADH. Increased lactate or lactate:pyruvate ratios have been measured in the blood, urine, and cerebrospinal fluid of a large number of Leigh syndrome patients (15, 16). Increases in other organic acids in urine have also been reported (16), indicating that metabolic acidosis is a prominent clinical feature. Interestingly, a study designed to find new diagnostic metabolites in MD demonstrated that within certain age ranges the measurement of urinary fumarate and malate was a more useful discriminator of MD than lactate or other organic acids (29). Barshop''s findings support the hypothesis that MD derived from OXPHOS deficiencies may exhibit increased protein succination because of the accumulation of NADH and subsequently fumarate. In this study we report for the first time that protein succination is present in the brain in an animal model of Leigh syndrome, the Ndufs4 KO mouse, suggesting that this modification may be an important biochemical link between the genetic defect and the onset of neuropathology observed in Leigh syndrome.     

Regulation of Myelin Basic Protein (Arginine) Methyltransferase by Thyroid Hormone in Myelinogenic Cultures of Cells Dissociated from Embryonic Mouse Brain

Abstract: The ontogenetic expression of myelin basic protein (arginine) methyltransferase in myelinogenic cultures of cells dissociated from embryonic mouse brain is highly dependent on the presence of thyroid hormone. Restoration of myelin basic protein methyltransferase to normal activities occurred 16 h after the addition of 100 n M l -3,5,3'-triiodothyronine to hypothyroid medium. These data demonstrate that thyroid hormone can regulate a posttranslational event. On the other hand, histone (arginine) methyltransferase has a different temporal activity pattern, which is not coordinated with myelination, and is not influenced by the lack of thyroid hormone. These data, which suggest the existence of two methyltransferases, were substantiated by demonstrating that the total amount of methylation of added myelin basic protein and histone is the same whether they are incubated together or separately. The requirement of thyroid hormone for the expression of the myelin basic protein methyltransferase and not for histone methyltransferase suggests that thyroid hormone preferentially regulates myelin-associated events in these cultures.     

Direct Comparison of the Efficacy and Safety of Oral Treatments with Oleylphosphocholine (OlPC) and Miltefosine in a Mouse Model of L. major Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis (CL) represents a range of skin diseases caused by infection with Leishmania parasites and associated with tissue inflammation and skin ulceration. CL is clinically widespread in both the Old and New World but lacks treatments that are well tolerated, effective and inexpensive. Oleylphosphocholine (OlPC) is a new orally bioavailable drug of the alkylphosphocholine family with potent antileishmanial activity against a broad range of Leishmania species/strains.

Methodology/principal findings

The potential of OlPC against Old World CL was evaluated in a mouse model of Leishmania (L.) major infection in BALB/c mice. Initial dose-response experiments showed that an oral daily dose of 40 mg/kg of OlPC was needed to impact time to cure and lesion sizes. This dose was then used to directly compare the efficacy of OlPC to the efficacy of the antileishmanial drugs miltefosine (40 mg/kg/day), fluconazole (160 mg/kg/day) and amphotericin B (25 mg/kg/day). OlPC, miltefosine and fluconazole were given orally for 21 days while amphotericin B was administered intraperitoneally for 10 days. Ulcer sizes and animal weights were followed up on a weekly basis and parasitemia was determined by means of a real-time in vivo imaging system which detects luminescence emitted from luciferase-expressing infecting L. major parasites. Amphotericin B and OlPC showed excellent efficacy against L. major lesions in terms of reduction of parasitic loads and by inducing complete healing of established lesions. In contrast, treatment with miltefosine did not significantly affect parasitemia and lesion sizes, while fluconazole was completely ineffective at the dose regimen tested.

Conclusions/Significance

Given the data showing the outstanding efficacy and tolerability of OlPC, our results suggest that OlPC is a promising new drug candidate to improve and simplify current clinical management of L. major CL.     

Calcium-dependent Activator Protein for Secretion 1 (CAPS1) Binds to Syntaxin-1 in a Distinct Mode from Munc13-1

Calcium-dependent activator protein for secretion 1 (CAPS1) is a multidomain protein containing a Munc13 homology domain 1 (MHD1). Although CAPS1 and Munc13-1 play crucial roles in the priming stage of secretion, their functions are non-redundant. Similar to Munc13-1, CAPS1 binds to syntaxin-1, a key t-SNARE protein in neurosecretion. However, whether CAPS1 interacts with syntaxin-1 in a similar mode to Munc13-1 remains unclear. Here, using yeast two-hybrid assays followed by biochemical binding experiments, we show that the region in CAPS1 consisting of the C-terminal half of the MHD1 with the corresponding C-terminal region can bind to syntaxin-1. Importantly, the binding mode of CAPS1 to syntaxin-1 is distinct from that of Munc13-1; CAPS1 binds to the full-length of cytoplasmic syntaxin-1 with preference to its “open” conformation, whereas Munc13-1 binds to the first 80 N-terminal residues of syntaxin-1. Unexpectedly, the majority of the MHD1 of CAPS1 is dispensable, whereas the C-terminal 69 residues are crucial for the binding to syntaxin-1. Functionally, a C-terminal truncation of 69 or 134 residues in CAPS1 abolishes its ability to reconstitute secretion in permeabilized PC12 cells. Our results reveal a novel mode of binding between CAPS1 and syntaxin-1, which play a crucial role in neurosecretion. We suggest that the distinct binding modes between CAPS1 and Munc13-1 can account for their non-redundant functions in neurosecretion. We also propose that the preferential binding of CAPS1 to open syntaxin-1 can contribute to the stabilization of the open state of syntaxin-1 during its transition from “closed” state to the SNARE complex formation.     

Selective Enhancement of Donor Hematopoietic Cell Engraftment by the CXCR4 Antagonist AMD3100 in a Mouse Transplantation Model

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.     

转基因水稻表达的Bt蛋白对拟环纹豹蛛（Pardosa pseudoannulata）体内保护酶活性的影响

把取食24h的转Cry1Ab／Cry1Ac基因水稻叶片的稻纵卷叶螟Cnaphalocrocis medinalis（Guenée）幼虫,分别喂养拟环纹豹蛛3天、6天、9天、12天后,采用酶活力测定方法探究了转基因水稻表达的Bt蛋白对拟环纹豹蛛体内3种保护酶（SOD、POD和CAT）活性的影响。结果表明：就整体来看,实验期间处理组蜘蛛体内3种保护酶（SOD、POD和CAT）的活性均受到Bt蛋白的影响,且前期3种保护酶活性均表现不同程度的被抑制,明显低于对照组。在实验过程中,SOD活性随着处理时间的增加而逐渐增强,至处理第9天时达到最大值;同时处理组POD、CAT活性也随着处理时间的增加而增强,实验初期（前6天）均显著低于对照组,而至第9天则均高于对照组。由此可见,Bt基因在水稻体内所表达的Bt蛋白能够沿食物链传递至次级消费者,并且在一定程度上对次级消费者产生影响。     

The Gene Coding for the β-Chain of C4b-Binding Protein (C4BPB) Has Become a Pseudogene in the Mouse

C4BPβ is one of the two polypeptides that in humans compose the plasma glycoprotein C4b-binding protein (C4BP). C4BPβ binds the anticoagulant vitamin K-dependent protein S. Two, nonmutually exclusive, roles have been proposed for the C4BP-protein S interaction. It has been suggested to play a role in the control of the protein C anticoagulatory pathway. In addition, it may serve an important role in localizing C4BP to the surface of injured or activated cells. While the physiological significance of C4BP-protein S interaction is unclear, it has clinical relevance because elevated plasma levels of C4BP are associated with increased risk for thromboembolic disorders in humans, due to an inactivation of the protein C anticoagulatory pathway. Using a human C4BPβ cDNA probe, we have isolated and characterized a genomic DNA fragment that includes the murine C4BPB gene. Murine C4BPB is a single-copy gene that maps close to the C4BPA gene in chromosome 1. It contains two exons homologous to the exons coding for the SCR-1 and SCR-2 repeats of the human C4BPβ polypeptide chain. Sequence analysis of the C4BPB exons in the Mus musculus inbred strains CBA, Balb/c, and C57BL/6, in penbred Swiss mice, and in Mus spretus demonstrated the presence of two in-phase stop codons that are incompatible with the expression of a functional C4BPβ polypeptide. Thus, the characterization of the murine C4BPB gene documents the peculiar situation of a single-copy gene that is functional in humans but has become a pseudogene in the mouse. Interestingly, the loss of a functional C4BPB gene is a relatively recent event in the evolution of the mouse. In addition, our data indicate that this genetic change has been fixed in the mouse population, suggesting that those individuals lacking the C4BPβ polypeptide were conferred with some kind of selective advantage.     

Immunosuppressive Drugs Modulate the Replication of Hepatitis B Virus (HBV) in a Hydrodynamic Injection Mouse Model

Hepatitis B virus (HBV) reactivation and recurrence are common in patients under immunosuppression and can be controlled by hepatitis B immunoglobulin, antivirals, and hepatitis B vaccine. However, the detailed analysis of HBV infection under immunosuppression is essential for the prophylaxis and therapy for HBV reactivation and recurrence. In this study, HBV replication and T cell responses were analyzed in a HBV-transfected mouse model under immunosuppressive therapy. During the treatment, HBV replication was at a high level in mice treated with dexamethasone, cyclosporine, and cyclophosphamide, whereas was terminated in mice treated with mycophenolate mofetil. After the withdrawal, HBV replication was at low or high levels in the dexamethasone-treated mice or in both cyclosporine- and cyclophosphamide-treated mice. The early withdrawal of cyclosporine allowed the recovery of suppressed T cell responses and led to subsequent HBV clearance, while the adoptive immune transfer to the mice with HBV persistence led to HBV suppression. Taken together, long-term HBV persistence under immunosuppression depends on the immunosuppressive drugs used and on the treatment duration and is mediated by the suppressed intrahepatic CD8 T cell response. These data may be helpful for individualized immunosuppressive therapy in patients with high risk of HBV reactivation and recurrence, and the mouse system is suitable for studying HBV reactivation and recurrence under immunosuppression.     

Metastatic Recurrence in a Pancreatic Cancer Patient Derived Orthotopic Xenograft (PDOX) Nude Mouse Model Is Inhibited by Neoadjuvant Chemotherapy in Combination with Fluorescence-Guided Surgery with an Anti-CA 19-9-Conjugated Fluorophore

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence.     

Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease.