Liraglutide, 7,8-DHF and their co-treatment prevents loss of vision and cognitive decline in a Wolfram syndrome rat model.

Wolfram syndrome (WS) is a monogenic progressive neurodegenerative disease and is characterized by various neurological symptoms, such as optic nerve atrophy, loss of vision, cognitive decline, memory impairment, and learning difficulties. GLP1 receptor agonist liraglutide and BDNF mimetic 7,8-dihydroxyflavone (7,8-DHF) have had protective effect to visual pathway and to learning and memory in different rat models of neurodegenerative disorders. Although synergistic co-treatment effect has not been reported before and therefore the aim of the current study was to investigate liraglutide, 7,8-DHF and most importantly for the first time their co-treatment effect on degenerative processes in WS rat model. We took 9 months old WS rats and their wild-type (WT) control animals and treated them daily with liraglutide, 7,8-DHF or with the combination of liraglutide and 7,8-DHF up to the age of 12.5 months (n = 47, 5-8 per group). We found that liraglutide, 7,8-DHF and their co-treatment all prevented lateral ventricle enlargement, improved learning in Morris Water maze, reduced neuronal inflammation, delayed the progression of optic nerve atrophy, had remyelinating effect on optic nerve and thereby improved visual acuity in WS rats compared to WT controls. Thus, the use of the liraglutide, 7,8-DHF and their co-treatment could potentially be used as a therapeutic intervention to induce neuroprotection or even neuronal regeneration.

GLP-1 receptor agonist liraglutide has a neuroprotective effect on an aged rat model of Wolfram syndrome.

Wolfram syndrome (WS) is a rare neurodegenerative disorder that is mainly characterized by diabetes mellitus, optic nerve atrophy, deafness, and progressive brainstem degeneration. Treatment with GLP-1 receptor agonists has shown a promising anti-diabetic effect in WS treatment in both animal models and in human patients. Since previous research has tended to focus on investigation of the WS first symptom, diabetes mellitus, the aim of the present study was to examine liraglutide effect on WS-associated neurodegeneration. We took 9-month-old Wfs1 knock-out (KO) animals that already had developed glucose intolerance and treated them with liraglutide for 6 months. Our research results indicate that 6-month liraglutide treatment reduced neuroinflammation and ameliorated endoplasmic reticulum (ER) stress in the inferior olive of the aged WS rat model. Liraglutide treatment also protected retinal ganglion cells from cell death and optic nerve axons from degeneration. According to this, the results of the present study provide novel insight that GLP-1 receptor agonist liraglutide has a neuroprotective effect in the WS rat model.

Moss Kinesin-14 KCBP Accelerates Chromatid Motility in Anaphase.

KCBP is a microtubule (MT) minus-end-directed kinesin widely conserved in plants. It was shown in Arabidopsis that KCBP controls trichome cell shape by orchestrating MT and actin cytoskeletons using its tail and motor domains. In contrast, the KCBP knockout (KO) line in the moss Physcomitrella patens showed a defect in nuclear and organelle positioning in apical stem cells. Moss KCBP is postulated to transport the nucleus and chloroplast via direct binding to their membranes, since it binds to and transports liposomes composed of phospholipids in vitro. However, domains required for cargo transport in vivo have not been mapped. Here, we performed a structure-function analysis of moss KCBP. We found that the FERM domain in the tail region, which is known to bind to lipids as well as other proteins, is essential for both nuclear and chloroplast positioning, whereas the proximal MyTH4 domain plays a supporting role in chloroplast transport. After anaphase but prior to nuclear envelope re-formation, KCBP accumulates on the chromosomes, in particular at the centromeric region in a FERM-dependent manner. In the KCBP KO line, the rate of poleward chromosome movement in anaphase was reduced and lagging chromosomes occasionally appeared. These results suggest that KCBP binds to non-membranous naked chromosomes via an unidentified protein(s) for their transport. Finally, the liverwort orthologue of KCBP rescued the chromosome/chloroplast mis-positioning of the moss KCBP KO line, suggesting that the cargo transport function is conserved at least in bryophytes.Key words: kinesin, mitosis, chromosome segregation, kinetochore, dynein.

A homozygous KAT2B variant modulates the clinical phenotype of ADD3 deficiency in humans and flies.

Recent evidence suggests that the presence of more than one pathogenic mutation in a single patient is more common than previously anticipated. One of the challenges hereby is to dissect the contribution of each gene mutation, for which animal models such as Drosophila can provide a valuable aid. Here, we identified three families with mutations in ADD3, encoding for adducin-γ, with intellectual disability, microcephaly, cataracts and skeletal defects. In one of the families with additional cardiomyopathy and steroid-resistant nephrotic syndrome (SRNS), we found a homozygous variant in KAT2B, encoding the lysine acetyltransferase 2B, with impact on KAT2B protein levels in patient fibroblasts, suggesting that this second mutation might contribute to the increased disease spectrum. In order to define the contribution of ADD3 and KAT2B mutations for the patient phenotype, we performed functional experiments in the Drosophila model. We found that both mutations were unable to fully rescue the viability of the respective null mutants of the Drosophila homologs, hts and Gcn5, suggesting that they are indeed pathogenic in flies. While the KAT2B/Gcn5 mutation additionally showed a significantly reduced ability to rescue morphological and functional defects of cardiomyocytes and nephrocytes (podocyte-like cells), this was not the case for the ADD3 mutant rescue. Yet, the simultaneous knockdown of KAT2B and ADD3 synergistically impaired kidney and heart function in flies as well as the adhesion and migration capacity of cultured human podocytes, indicating that mutations in both genes may be required for the full clinical manifestation. Altogether, our studies describe the expansion of the phenotypic spectrum in ADD3 deficiency associated with a homozygous likely pathogenic KAT2B variant and thereby identify KAT2B as a susceptibility gene for kidney and heart disease in ADD3-associated disorders.

Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size.

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability) 1 , studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2-4, but have little effect on the brains of mice5-9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12-14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25-40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of 'cortical units' has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12-16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.

Rescue of cone function in cone-only Nphp5 knockout mouse model with Leber congenital amaurosis phenotype.

Purpose: Recessive mutations in the human IQCB1/NPHP5 gene are associated with Senior-Løken syndrome (SLS), a ciliopathy presenting with nephronophthisis and Leber congenital amaurosis (LCA). Nphp5-knockout mice develop LCA without nephronophthisis. Mutant rods rapidly degenerate while mutant cones survive for months. The purpose of this study was to reinitiate cone ciliogenesis in a Nphp5 -/-; Nrl -/- mouse with viral expression of full-length NPHP5 and rescue function. Methods: Nphp5 -/- mice were mated with Nrl -/- mice to generate Nphp5-/-; Nrl-/- double-knockouts. Nphp5-/-; Nrl-/- mice and Nphp5+/-; Nrl-/- controls were phenotyped with confocal microscopy from postnatal day 10 (P10) until 6 months of age. Nphp5-/-; Nrl-/- mice and Nphp5+/-; Nrl-/- controls were injected at P15 with self-complementary adenoassociated virus 8 (Y733F) (AAV8(Y733F)) expressing GRK1-FL-cNPHP5. Expression of mutant NPHP5 was verified with confocal microscopy and electroretinography (ERG). Results: In the Nphp5 -/- and cone-only Nphp5 -/-; Nrl -/- mice, cone outer segments did not form, but mutant cones continued to express cone pigments in the inner segments without obvious signs of cone cell death. The mutant cone outer nuclear layer (ONL) and the inner segments were stable for more than 6 months in the cone-only Nphp5 -/-; Nrl -/- retinas. Viral expression of NPHP5 initiated after eye opening showed that connecting cilia and RP1-positive axonemes were formed. Furthermore, cone pigments and other cone outer segment proteins (cone transducin and cone PDE6) were present in the nascent mutant cone outer segments, and rescued mutant cones exhibited a significant photopic b-wave (30% of Nphp5 +/-; Nrl -/- controls). Conclusions: Nphp5-/-; Nrl-/- cones persistently express cone pigments in the inner segments without obvious degeneration, providing an extended duration interval for viral gene expression. Viral expression of full-length NPHP5 initiates ciliogenesis between P15 and P60, and mutant cones are, in part, functional, encouraging future retina gene replacement therapy.

Altered synaptic phospholipid signaling in PRG-1 deficient mice induces exploratory behavior and motor hyperactivity resembling psychiatric disorders.

Plasticity related gene 1 (PRG-1) is a neuron specific membrane protein located at the postsynaptic density of glutamatergic synapses. PRG-1 modulates signaling pathways of phosphorylated lipid substrates such as lysophosphatidic acid (LPA). Deletion of PRG-1 increases presynaptic glutamate release probability leading to neuronal over-excitation. However, due to its cortical expression, PRG-1 deficiency leading to increased glutamatergic transmission is supposed to also affect motor pathways. We therefore analyzed the effects of PRG-1 function on exploratory and motor behavior using homozygous PRG-1 knockout (PRG-1-/-) mice and PRG-1/LPA2-receptor double knockout (PRG-1-/-/LPA2-/-) mice in two open field settings of different size and assessing motor behavior in the Rota Rod test. PRG-1-/- mice displayed significantly longer path lengths and higher running speed in both open field conditions. In addition, PRG-1-/- mice spent significantly longer time in the larger open field and displayed rearing and self-grooming behavior. Furthermore PRG-1-/- mice displayed stereotypical behavior resembling phenotypes of psychiatric disorders in the smaller sized open field arena. Altogether, this behavior is similar to the stereotypical behavior observed in animal models for psychiatric disease of autistic spectrum disorders which reflects a disrupted balance between glutamatergic and GABAergic synapses. These differences indicate an altered excitation/inhibition balance in neuronal circuits in PRG-1-/- mice as recently shown in the somatosensory cortex [38]. In contrast, PRG-1-/-/LPA2-/- did not show significant changes in behavior in the open field suggesting that these specific alterations were abolished when the LPA2-receptor was lacking. Our findings indicate that PRG-1 deficiency led to over-excitability caused by an altered LPA/LPA2-R signaling inducing a behavioral phenotype typically observed in animal models for psychiatric disorders.

Longitudinal Diffusion Tensor Imaging Revealed Nerve Fiber Alterations in Aspm Mutated Microcephaly Model Mice.

Autosomal recessive primary microcephaly-5 (MCPH5) is characterized by congenital microcephaly and is caused by the mutation in the abnormal spindle-like, microcephaly-associated (ASPM) gene. This study aimed to demonstrate a correlation between radiological and pathological analyses in evaluating postnatal brain development using MCPH5-model mice, ASPM ortholog (Aspm) knockout (KO) mice. In vivo MRI was performed at two time points (postnatal 3 weeks; P3W and P10W) and complementary histopathological analyses of brains were done at P5W and P13W. In the MRI analysis, Aspm KO mice showed significantly decreased brain sizes (average 8.6% difference) with larger ventricles (average 136.4% difference) at both time points. Voxel-based statistics showed that the fractional anisotropy (FA) values were significantly lower in Aspm KO mice in both the cortex and white matter at both time points. Developmental changes in the FA values were less remarkable in the Aspm KO mice, compared with the controls. Histometric analyses revealed that the ratios of the horizontal to the vertical neurites were significantly higher in cortical layers IV, V and VI, with a remarkable increase according to maturation at P13W in the control mice (average 12.7% difference between control and KO), whereas the ratio in layer VI decreased at P13W in the KO mice. The myelin basic protein positive ratio in the white matter significantly decreased in Aspm KO mice at P5W. These results suggest that temporal FA changes are closely correlated with pathological findings such as abnormal neurite outgrowth and differentiation, which may be applicable for analyzing diseased human brain development.

Integrated analysis of omics data using microRNA-target mRNA network and PPI network reveals regulation of Gnai1 function in the spinal cord of Ews/Ewsr1 KO mice.

BACKGROUND: Multifunctional transcription factor (TF) gene EWS/EWSR1 is involved in various cellular processes such as transcription regulation, noncoding RNA regulation, splicing regulation, genotoxic stress response, and cancer generation. Role of a TF gene can be effectively studied by measuring genome-wide gene expression, i.e., transcriptome, in an animal model of Ews/Ewsr1 knockout (KO). However, when a TF gene has complex multi-functions, conventional approaches such as differentially expressed genes (DEGs) analysis are not successful to characterize the role of the EWS gene. In this regard, network-based analyses that consider associations among genes are the most promising approach. METHODS: Networks are constructed and used to show associations among biological entities at various levels, thus different networks represent association at different levels. Taken together, in this paper, we report contributions on both computational and biological sides. RESULTS: Contribution on the computational side is to develop a novel computational framework that combines miRNA-gene network and protein-protein interaction network information to characterize the multifunctional role of EWS gene. On the biological side, we report that EWS regulates G-protein, Gnai1, in the spinal cord of Ews/Ewsr1 KO mice using the two biological network integrated analysis method. Neighbor proteins of Gnai1, G-protein complex subunits Gnb1, Gnb2 and Gnb4 were also down-regulated at their gene expression level. Interestingly, up-regulated genes, such as Rgs1 and Rgs19, are linked to the inhibition of Gnai1 activities. We further verified the altered expression of Gnai1 by qRT-PCR in Ews/Ewsr1 KO mice. CONCLUSIONS: Our integrated analysis of miRNA-transcriptome network and PPI network combined with qRT-PCR verifies that Gnai1 function is impaired in the spinal cord of Ews/Ewsr1 KO mice.

Modulation of autophagy by miRNAs.

MicroRNAs (miRNAs) can regulate the expression of genes that are involved in multiple cellular pathways. However, their targets and mechanism of action associated with the autophagy pathway are not fully investigated yet. EWSR1 (EWS RNA-Binding Protein 1/Ewing Sarcoma Break Point Region 1) gene encodes a RNA/DNA binding protein that is ubiquitously expressed and plays roles in numerous cellular processes. Recently, our group has shown that EWSR1 deficiency leads to developmental failure and accelerated senescence via processing of miRNAs, but its role in the regulation of autophagy remains elusive. In this context, we further investigated and found that EWSR1 deficiency triggers the activation of the DROSHA-mediated microprocessor complex and increases the levels of miR125a and miR351, which directly target Uvrag. Interestingly, the miR125a- and miR351-targeted reduction of Uvrag led to the inhibition of autophagy in both ewsr1 knockout (KO) MEFs and ewsr1 KO mice. In summary, our study demonstrates that EWSR1 is associated with the posttranscriptional regulation of Uvrag via miRNA processing. The regulation of autophagy pathway in miRNAs-Uvrag-dependent manner provides a novel mechanism of EWSR1 deficiency-related cellular dysfunction.

Uvrag targeting by Mir125a and Mir351 modulates autophagy associated with Ewsr1 deficiency.

The EWSR1 (EWS RNA-binding protein 1/Ewing Sarcoma Break Point Region 1) gene encodes a RNA/DNA binding protein that is ubiquitously expressed and involved in various cellular processes. EWSR1 deficiency leads to impairment of development and accelerated senescence but the mechanism is not known. Herein, we found that EWSR1 modulates the Uvrag (UV radiation resistance associated) gene at the post-transcription level. Interestingly, EWSR1 deficiency led to the activation of the DROSHA-mediated microprocessor complex and increased the level of Mir125a and Mir351, which directly target Uvrag. Moreover, the Mir125a- and Mir351-mediated reduction of Uvrag was associated with the inhibition of autophagy that was confirmed in ewsr1 knockout (KO) MEFs and ewsr1 KO mice. Taken together, our data indicate that EWSR1 is involved in the post-transcriptional regulation of Uvrag via a miRNA-dependent pathway, resulting in the deregulation of autophagy inhibition. The mechanism of Uvrag and autophagy regulation by EWSR1 provides new insights into the role of EWSR1 deficiency-related cellular dysfunction.

miR-34/449 miRNAs are required for motile ciliogenesis by repressing cp110.

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.

Iqcg is essential for sperm flagellum formation in mice.

Mammalian spermatogenesis comprises three successive phases: mitosis phase, meiosis phase, and spermiogenesis. During spermiogenesis, round spermatid undergoes dramatic morphogenesis to give rise to mature spermatozoon, including the condensation and elongation of nucleus, development of acrosome, formation of flagellum, and removal of excessive cytoplasm. Although these transformations are well defined at the morphological level, the mechanisms underlying these intricate processes are largely unknown. Here, we report that Iqcg, which was previously characterized to be involved in a chromosome translocation of human leukemia, is highly expressed in the spermatogenesis of mice and localized to the manchette in developing spermatids. Iqcg knockout causes male infertility, due to severe defects of spermiogenesis and resultant total immobility of spermatozoa. The axoneme in the Iqcg knockout sperm flagellum is disorganized and hardly any typical ("9+2") pattern of microtubule arrangement could be found in Iqcg knockout spermatids. Iqcg interacts with calmodulin in a calcium dependent manner in the testis, suggesting that Iqcg may play a role through calcium signaling. Furthermore, cilia structures in the trachea and oviduct, as well as histological appearances of other major tissues, remain unchanged in the Iqcg knockout mice, suggesting that Iqcg is specifically required for spermiogenesis in mammals. These results might also provide new insights into the genetic causes of human infertility.

Atomic force microscopy demonstration of cytoskeleton instability in mouse erythrocytes with dematin-headpiece and ß-adducin deficiency.

The pattern of disassembly of the cytoskeletal network of murine erythrocytes with deficiency of either dematin-headpiece or ß-adducin or both proteins were investigated using atomic force microscopy. A heterogeneous complex structure with fine filament features and coarse features was observed in the cytoskeleton of wild type mouse erythrocytes, whereas a significant amount of rearrangement and aggregation occurred in the mutants, particularly in the cells carrying double gene mutations. These results are consistent with the cellular and biochemical phenotype of the mutant cell membranes as being more fragile due to weakened vertical connections with the plasma membrane.

Comparative proteomics reveals deficiency of NHE-1 (Slc9a1) in RBCs from the beta-adducin knockout mouse model of hemolytic anemia.

Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium-hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice.

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells.

Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

beta-adducin (Add2) KO mice show synaptic plasticity, motor coordination and behavioral deficits accompanied by changes in the expression and phosphorylation levels of the alpha- and gamma-adducin subunits.

Adducins are a family of proteins found in cytoskeleton junctional complexes, which bind and regulate actin filaments and actin-spectrin complexes. In brain, adducin is expressed at high levels and is identified as a constituent of synaptic structures, such as dendritic spines and growth cones of neurons. Adducin-induced changes in dendritic spines are involved in activity-dependent synaptic plasticity processes associated with learning and memory, but the mechanisms underlying these functions remain to be elucidated. Here, beta-adducin knockout (KO) mice were used to obtain a deeper insight into the role of adducin in these processes. We showed that beta-adducin KO mice showed behavioral, motor coordination and learning deficits together with an altered expression and/or phosphorylation levels of alpha-adducin and gamma-adducin. We found that beta-adducin KO mice exhibited deficits in learning and motor performances associated with an impairment of long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. These effects were accompanied by a decrease in phosphorylation of adducin, a reduction in alpha-adducin expression levels and upregulation of gamma-adducin in hippocampus, cerebellum and neocortex of mutant mice. In addition, we found that the mRNA encoding beta-adducin is also located in dendrites, where it may participate in the fine modulation of LTP and LTD. These results strongly suggest coordinated expression and phosphorylation of adducin subunits as a key mechanism underlying synaptic plasticity, motor coordination performance and learning behaviors.

The membrane cytoskeletal protein adducin is phosphorylated by protein kinase C in D1 neurons of the nucleus accumbens and dorsal striatum following cocaine administration.

Repeated cocaine administration results in persistent changes in synaptic function in the mesolimbic dopamine system that are thought to be critical for the transition to addiction. Cytoskeletal rearrangement and actin dynamics are essential for this drug-dependent plasticity. Cocaine administration increases levels of F-actin in the nucleus accumbens and is associated with changes in the phosphorylation state of actin-binding proteins. The adducins constitute a family of proteins that interact with actin and spectrin to maintain cellular architecture. The interaction of adducin with these cytoskeletal proteins is regulated by phosphorylation, and it is therefore expected that phosphorylation of adducin may be involved in morphological changes underlying synaptic responses to drugs of abuse including cocaine. In the current study, we characterized the regulation of adducin phosphorylation in the nucleus accumbens and dorsal striatum in response to various regimen of cocaine. Our results demonstrate that adducin is phosphorylated by protein kinase C in medium spiny neurons that express the dopamine D1 receptor. These data indicate that adducin phosphorylation is a signaling event regulated by cocaine administration and further suggest that adducin may be involved in remodeling of the neuronal cytoskeleton in response to cocaine administration.

Targeted deletion of the gamma-adducin gene (Add3) in mice reveals differences in alpha-adducin interactions in erythroid and nonerythroid cells.

In red blood cells (RBCs) adducin heterotetramers localize to the spectrin-actin junction of the peripheral membrane skeleton. We previously reported that deletion of beta-adducin results in osmotically fragile, microcytic RBCs and a phenotype of hereditary spherocytosis (HS). Notably, alpha-adducin was significantly reduced, while gamma-adducin, normally present in limited amounts, was increased approximately 5-fold, suggesting that alpha-adducin requires a heterologous binding partner for stability and function, and that gamma-adducin can partially substitute for the absence of beta-adducin. To test these assumptions we generated gamma-adducin null mice. gamma-adducin null RBCs appear normal on Wright's stained peripheral blood smears and by scanning electron microscopy. All membrane skeleton proteins examined are present in normal amounts, and all hematological parameters measured are normal. Despite a loss of approximately 70% of alpha-adducin in gamma-adducin null platelets, no bleeding defect is observed and platelet structure appears normal. Moreover, systemic blood pressure and pulse are normal in gamma-adducin null mice. gamma- and beta-adducin null mice were intercrossed to generate double null mice. Loss of gamma-adducin does not exacerbate the beta-adducin null HS phenotype although the amount alpha-adducin is reduced to barely detectable levels. The stability of alpha-adducin in the absence of a heterologous binding partner varies considerably in various tissues. The amount of alpha-adducin is modestly reduced ( approximately 15%) in the kidney, while in the spleen and brain is reduced by approximately 50% with the loss of a heterologous beta- or gamma-adducin binding partner. These results suggest that the structural properties of adducin differ significantly between erythroid and various nonerythroid cell types.