Young Knockout Mice Research Articles

Vasoactive intestinal peptide deficiency promotes ovarian dysfunction associated to a proinflammatory microenvironment reminiscent of premature aging

Complex immune regulation during pregnancy is required to ensure a successful pregnancy outcome. Vasoactive intestinal peptide (VIP) has local immunoregulatory effects on the ovary, uterus and maternal-fetal interface that favor a tolerogenic maternal microenvironment. Since the VIP Knockout (KO) mice are subfertile, we investigated the mechanisms underlying the effects of VIP deficiency on ovarian physiology and immune homeostasis. Therefore, we studied VIP KO, deficient (HT) and wild type (WT) female mice in estrus at 3 or 8 months of age. Young KO mice showed abnormal cycle timing and regularity associated with dysfunctional ovaries. Ovaries presented higher number of atretic follicles and reduced number of corpora lutea leading to a lower ovulation rates. Part of the VIP KO mice (25 %) failed to ovulate or ovulated oocytes incompetent to be fertilized (50 %). In particular, ovaries of young KO mice exhibited features of premature aging accompanied by a pro-inflammatory milieu with increased levels of IL-1β. A unique macrophage subpopulation identified as “foamy macrophages” was found. On the other hand, aged VIP KO females did not gain body weight probably due to the sustained production of E2. Finally, the adoptive transfer of FOXP3+ cells to infertile VIP KO females resulted in their selective recruitment to the ovary. It increased FOXP3/RORγt and TGFβ/IL-6 ratio improving ovarian microenvironment and pregnancy rate. The present results suggest that VIP contributes to ovarian homeostatic mechanisms required for a successful pregnancy.

Developmental oxidative stress leads to T-type Ca2+ channel hypofunction in thalamic reticular nucleus of mouse models pertinent to schizophrenia

Impairment of parvalbumin interneurons induced by oxidative stress (OxS) is a “hub” on which converge several genetic and environmental risk factors associated with schizophrenia. In patients, this could be a mechanism leading to anomalies of the thalamic reticular nucleus (TRN) whose major neuronal population expresses parvalbumin. The TRN shapes the information flow within thalamo-cortical circuits. The low-threshold voltage-gated T-type Ca2+ (T-Ca2+) channels (CaV3.2, CaV3.3) contribute to the excitability and rhythmic bursting of TRN neurons which mediates cortical sleep spindles, known to be affected in schizophrenia. Here, we investigated the impact of OxS during postnatal development and adulthood on firing properties and T-Ca2+ channels of TRN neurons. In Gclm knock-out (KO) mice, which display GSH deficit and OxS in TRN, we found a reduction of T-Ca2+ current density in adulthood, but not at peripuberty. In KO adults, the decreased T-Ca2+ currents were accompanied with a decrease of CaV3.3 expression, and a shift towards more hyperpolarized membrane potentials for burst firing leading to less prominent bursting profile. In young KO mice, an early-life oxidative challenge precipitated the hypofunction of T-Ca2+ channels. This was prevented by a treatment with N-acetylcysteine. The concomitant presence of OxS and hypofunction of T-Ca2+ channels were also observed in TRN of a neurodevelopmental model relevant to psychosis (MAM mice). Collectively, these data indicate that OxS-mediated T-Ca2+ hypofunction in TRN begins early in life. This also points to T-Ca2+ channels as one target of antioxidant-based treatments aiming to mitigate abnormal thalamo-cortical communication and pathogenesis of schizophrenia.

MiR-15a/16-1 deficiency induces IL-10-producing CD19+ TIM-1+ cells in tumor microenvironment.

IL‐10‐producing B cells (B10) are associated with autoimmune diseases, infection and tumours. MiR‐15a/16 as a tumour‐suppressive gene is down‐regulated in several tumours, such as chronic lymphocytic leukaemia, pituitary adenomas and prostate carcinoma. Here, increased frequency of IL‐10‐producing CD19+ Tim‐1+ cells was seen in both aged miR‐15a/16−/− mice (15‐18 months) with the onset of B cell leukaemia and young knockout mice (8‐12 weeks) transplanted with hepatic cancer cells. CD19+ Tim‐1+ cells down‐regulated the function of effector CD4+CD25low T cells ex vivo dependent on IL‐10 production, and adoptive transfer of CD19+ Tim‐1+ cells promoted tumour growth in mice. IL‐10 production by CD19+ Tim‐1+ cells was involved with the STAT3 activation. Bioinformatics analysis shows that miR‐16 targets the 3′‐untranslating region (3′‐UTR) of STAT3 mRNA. Overexpression of miR‐16 in CD19+ Tim‐1+ cells inhibited STAT3 transcription and its protein expression. Thus, the loss of miR‐15a/16 promoted induction of regulatory CD19+ Tim‐1+ cells in tumour microenvironment. These results confirmed that miR‐15a/16 could be used in tumour therapy due to its inhibition of tumour and regulatory B cells.

Tamm-Horsfall protein/uromodulin deficiency elicits tubular compensatory responses leading to hypertension and hyperuricemia.

Expression of Tamm-Horsfall protein (THP or uromodulin) is highly restricted to the kidney thick ascending limb (TAL) of loop of Henle. Despite the unique location and recent association of THP gene mutations with hereditary uromodulin-associated kidney disease and THP single nucleotide polymorphisms with chronic kidney disease and hypertension, the physiological function(s) of THP and its pathological involvement remain incompletely understood. By studying age-dependent changes of THP knockout (KO) mice, we show here that young KO mice had significant salt and water wasting but were partially responsive to furosemide, due to decreased luminal translocation of Na-K-Cl cotransporter 2 (NKCC2) in the TAL. Aged THP KO mice were, however, markedly oliguric and unresponsive to furosemide, and their NKCC2 was localized primarily in the cytoplasm as evidenced by lipid raft floatation assay, cell fractionation, and confocal and immunoelectron microscopy. These aged KO mice responded to metolazone and acetazolamide, known to target distal and proximal tubules, respectively. They also had marked upregulation of renin in juxtaglomerular apparatus and serum, and they were hypertensive. Finally, the aged THP KO mice had significant upregulation of Na-coupled urate transporters Slc5a8 and Slc22a12 as well as sodium-hydrogen exchanger 3 (NHE3) in the proximal tubule and elevated serum uric acid and allantoin. Collectively, our results suggest that THP deficiency can cause progressive disturbances in renal functions via initially NKCC2 dysfunction and later compensatory responses, resulting in prolonged activation of the renin-angiotensin-aldosterone axis and hyperuricemia.

Autophagy and mitochondrial biogenesis impairment contribute to age‐dependent liver injury in experimental sepsis: dysregulation of AMP‐activated protein kinase pathway

Age is an independent risk factor of multiple organ failure in patients with sepsis. However, the age-related mechanisms of injury are not known. AMPK is a crucial regulator of energy homeostasis, which controls mitochondrial biogenesis by activation of peroxisome proliferator-activated receptor-γ coactivator-α (PGC-1α) and disposal of defective organelles by autophagy. We investigated whether AMPK dysregulation might contribute to age-dependent liver injury in young (2-3 mo) and mature male mice (11-13 mo) subjected to sepsis. Liver damage was higher in mature mice than in young mice and was associated with impairment of hepatocyte mitochondrial function, structure, and biogenesis and reduced autophagy. At molecular analysis, there was a time-dependent nuclear translocation of the active phosphorylated catalytic subunits AMPKα1/α2 and PGC-1α in young, but not in mature, mice after sepsis. Treatment with the AMPK activator 5-amino-4-imidazolecarboxamide riboside-1-β-d-ribofuranoside (AICAR) improved liver mitochondrial structure in both age groups compared with vehicle. In loss-of-function studies, young knockout mice with systemic deficiency of AMPKα1 exhibited greater liver injury than did wild-type mice after sepsis. Our study suggests that AMPK is important for liver metabolic recovery during sepsis. Although its function may diminish with age, pharmacological activation of AMPK may be of therapeutic benefit.-Inata, Y., Kikuchi, S., Samraj, R. S., Hake, P. W., O'Connor, M., Ledford, J. R., O'Connor, J., Lahni, P., Wolfe, V., Piraino, G., Zingarelli, B. Autophagy and mitochondrial biogenesis impairment contribute to age-dependent liver injury in experimental sepsis: dysregulation of AMP-activated protein kinase pathway.

P 088 - p38α and NF-κB regulate antioxidant defense in the liver through an age-dependent mechanism

p38α MAPK is a sensor of oxidative stress. The aim of this work was to assess the role of p38α in the regulation of the antioxidant defense in the liver with aging. Livers ofyoung and old wild type (WT) and p38α liver-specific knock out (KO) mice were used to determine glutathione redox status by mass spectrometry; malondialdehyde (MDA) levels by HPLC; mRNA expression of glutamate cysteine ligase (Gclc), Sod1, Sod2 and catalase by RT-PCR and nuclear levels of NF-κB subunit p65 by western-blotting. Chromatin immunoprecipitation (ChIP) assay of p65 was performed. Young KO liver exhibited increased in GSSG/GSH ratio and MDA levels when are compared with young WT mice. However, old KO mice had lower hepatic GSSG/GSH ratio and MDA levels than young KO mice. The mRNA expression of Gclc, Sod1, Sod2 and catalase was decreased in young KO mice compared with WT young mice. In contrast, these antioxidant enzymes were up-regulated in KO old mice regard to WT. p65 was markedly phosphorylated in the nucleus of WT young and old KO mice. ChIP assay of p65 confirmed the recruitment of p65 to the promoters of antioxidant genes in WT young and KO old mice. Deficiency of p38α in the liver causes dramatic down-regulation of antioxidant enzymes in young mice, but triggers adaptive up-regulation in the long term via NF-κB to enhance the antioxidant defense in old p38α KO mice.

188 - Peroxiredoxin-5 as a Novel Actor in Inflammation and Tumor Suppression

Peroxiredoxins are a superfamily of peroxidases widely distributed among prokaryotes and eukaryotes. Peroxiredoxin-5 (Prdx5) is the last isoform identified and characterized among the six mammalian peroxiredoxins. Prdx5 is ubiquitously expressed in tissues and its role as cytoprotective antioxidant enzyme has been largely reported in vitro. The goal of this work was to study Prdx5 function in vivo through the generation of transgenic mice in which Prdx5 gene has been disrupted using a gene-trap strategy. Prdx5 knockout (KO) mice are viable and fertile. Young KO mice do not suffer from any apparent defects. Behavioral tests and complete histological analyses of adult Prdx5 KO mice did not reveal major abnormalities. Microarray-based mRNA profiles of KO and WT mice showed that several CYP450 genes were highly upregulated in liver and brain of KO mice. Because Prdx5 gene has been shown to be upregulated in mouse macrophages upon pro-inflammatory stimulation, the vulnerability of Prdx5 KO mice to lipopolysaccharide (LPS) was examined. Our results show that Prdx5 KO mice mortality is higher after intraperitoneal injection of 20mg/kg and 10mg/kg of LPS, suggesting that Prdx5 may play key roles in inflammation either by protecting cells against oxidative damages by modulating inflammatory redox signaling or acting as damage-associated molecular pattern molecule. Finally, after 14 months, 20% of Prdx5 KO mice developed malignant cancers, mainly lymphoma, suggesting that Prdx5 may also function as a tumor suppressor.

Rhox13 is required for a quantitatively normal first wave of spermatogenesis in mice.

We previously described a novel germ cell-specific X-linked reproductive homeobox gene (Rhox13) that is upregulated at the level of translation in response to retinoic acid (RA) in differentiating spermatogonia and preleptotene spermatocytes. We hypothesize that RHOX13 plays an essential role in male germ cell differentiation, and have tested this by creating a Rhox13 gene knockout (KO) mouse. Rhox13 KO mice are born in expected Mendelian ratios, and adults have slightly reduced testis weights, yet a full complement of spermatogenic cell types. Young KO mice (at ~7-8 weeks of age) have a ≈50% reduction in epididymal sperm counts, but numbers increased to WT levels as the mice reach ~17 weeks of age. Histological analysis of testes from juvenile KO mice reveals a number of defects during the first wave of spermatogenesis. These include increased apoptosis, delayed appearance of round spermatids and disruption of the precise stage-specific association of germ cells within the seminiferous tubules. Breeding studies reveal that both young and aged KO males produce normal-sized litters. Taken together, our results indicate that RHOX13 is not essential for mouse fertility in a controlled laboratory setting, but that it is required for optimal development of differentiating germ cells and progression of the first wave of spermatogenesis.

Butyrylcholinesterase Deficiency Promotes Adipose Tissue Growth and Hepatic Lipid Accumulation in Male Mice on High-Fat Diet.

Despite numerous reports of relationships between weight gain and butyrylcholinesterase (BChE), this enzyme's role in the genesis of obesity remains unclear, but recent research points to strong links with ghrelin, the "hunger hormone." The availability of BChE knockout (KO) mice provides an opportunity to clarify the causal relationship between BChE and obesity onset. We now find that young KO mice have abnormally high plasma ghrelin levels that slowly decline during long-term high-fat feeding and ultimately drop below those in wild-type mice. On such a diet, the KO mice gained notably more weight, more white fat, and more hepatic fat than wild-type animals. In addition to a greater burden of hepatic triglycerides, the livers of these KO mice show distinctly higher levels of inflammatory markers. Finally, their energy expenditure proved to be lower than in wild-type mice despite similar activity levels and increased caloric intake. A gene transfer of mouse BChE with adeno-associated virus vector restored nearly all aspects of the normal phenotype. Our results indicate that BChE strongly affects fat metabolism, has an important impact on fat accumulation, and may be a promising tool for combating obesity.

Effect of systemic high dose enzyme replacement therapy on the improvement of CNS defects in a mouse model of mucopolysaccharidosis type II.

BackgroundMucopolysaccharidosis type II (MPS II, Hunter syndrome), is caused by a deficiency of iduronate-2-sulfatase (IDS). Despite the therapeutic effect of intravenous enzyme replacement therapy (ERT), the central nervous system (CNS) defects persist because the enzyme cannot cross the blood-brain barrier (BBB). There have been several trials of direct infusion to the cerebrospinal space showing promising results; however, this approach may have limitations in clinical situations such as CNS infection. The objective of this study was to improve the CNS defect with systemic high-dose ERT.MethodsSystemic ERT was performed using three doses (1, 5, and 10 mg/kg weekly) of IDS for three different durations (1, 3, and 6 months) in IDS knock out (KO) mice of two age groups (2 months, 8 months). GAG measurement in tissues, brain pathology, and behavioral assessment were analyzed.ResultsBrain IDS activities increased in parallel with the concentrations of IDS injected. The glycosaminoglycan (GAG) level and histopathology in the brains of the young mice improved in a dose- and duration-dependent manner; however, those were not improved in the old mice, even at higher doses of IDS. The spontaneous alternation behavior was recovered in young KO mice treated with ≥ 5 mg/kg IDS; however, no significant improvement was observed in old KO mice.ConclusionsThese results suggest that high-dose ERT given to mice of earlier ages may play a role in preventing GAG accumulation and preventing CNS damage in IDS KO mice. Therefore, ERT above the present standard dose, starting in early childhood, could be a promising treatment regimen for reducing neurological impairment in Hunter syndrome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0356-0) contains supplementary material, which is available to authorized users.

Deletion of the Insulin Receptor in the Proximal Tubule Promotes Hyperglycemia

Nearly all renal tubular epithelial cells express insulin receptor. The insulin receptor in the distal tubule appears to modulate BP, but the role of the insulin receptor in the proximal tubule is unknown. Here, we selectively knocked out the insulin receptor from the proximal tubules of mice. Western blotting confirmed a two- to three-fold reduction in renal cortical homogenate insulin receptor-β among knockout mice compared with wild-type littermates. Young knockout mice exhibited a mildly diabetic phenotype, evidenced by higher fasting plasma glucose levels than wild-type mice. Assessments by hyperinsulinemic-euglycemic clamp and a glucose tolerance test revealed no differences in insulin sensitivity or overt pancreatic function, respectively. Renal cortical mRNA expression and enzyme activity of glucose-6-phosphatase, which catalyzes the final step of glucose production, were significantly higher in knockout mice. Taken together, these results support a role for insulin receptor in the proximal tubule in the modulation of systemic glucose levels. Downregulation of the insulin receptor in the proximal tubule, which occurs in insulin-resistant states, may promote hyperglycemia through enhanced gluconeogenesis.

Status of muscle signaling pathways in the early pathogenesis of dystroglycanopathy‐type muscular dystrophy

A subset of muscular dystrophies are caused by abnormal glycosylation of α‐dystroglycan. Without its proper sugar structure, α‐dystroglycan binding to extracellular matrix proteins (e.g. laminin, perlecan) is compromised, disrupting the structural integrity of muscle and other tissues. Mutations in the FCMD gene, encoding fukutin, cause severe and milder dystroglycanopathies, Fukuyama congenital muscular dystrophy (FCMD) and limb‐girdle muscular dystrophy type 2M (LGMD 2M), respectively. Using fukutin conditional knockout mice, we have evidence that severe congenital muscular dystrophy phenotypes involve abnormal skeletal muscle development and/or differentiation. We will present a molecular, biochemical and histological approach, screening select muscle signaling pathways in young knockout mice, to identify abnormalities in skeletal muscle development/differentiation. These findings bring new insight to potential mechanisms underlying early disease pathogenesis and severe phenotypic outcomes in dystroglycanopathy‐type congenital muscular dystrophies.This work has been supported by the Muscular Dystrophy Association (Development Grant) and the University of Georgia, College of Pharmacy.

X-linked juvenile retinoschisis: clinical diagnosis, genetic analysis, and molecular mechanisms.

X-linked juvenile retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long-term retinoschisin expression and rescue of retinal structure and function providing a 'proof of concept' that gene therapy may be an effective treatment for XLRS.

The RANKL Distal Control Region Is Required for the Increase in RANKL Expression, But Not the Bone Loss, Associated with Hyperparathyroidism or Lactation in Adult Mice

Osteoclast-mediated bone resorption plays an essential role in calcium homeostasis and lactation. The cytokine receptor activator of nuclear factor κB ligand (RANKL) is one of a number of factors that controls the production, survival, and activity of osteoclasts. Calciotropic hormones, such as PTH, control RANKL transcription in part via an enhancer known as the distal control region (DCR), and mice lacking this enhancer have fewer osteoclasts under normal physiological conditions. Here, we have addressed the role of the DCR in situations in which activation of the PTH receptor is thought to stimulate bone resorption via elevation of RANKL expression. Dietary calcium deficiency stimulated RANKL expression in the bone of young (1 month old) wild-type, but not DCR knockout (KO), mice. Consistent with this, the cancellous bone loss and the increase in osteoclasts caused by dietary calcium deficiency were blunted in young KO mice. DCR deletion also prevented the increase in RANKL expression caused by dietary calcium deficiency in 6-month-old mice. However, the diet-induced bone loss was similar in wild-type and KO mice at this age. The increase in RANKL expression caused by lactation was also blunted in DCR KO mice, but lactation-induced bone loss was similar in both genotypes. These results demonstrate that, even though the DCR is required for the increase in RANKL expression associated with hyperparathyroidism or lactation, this increase is not required for the bone loss caused by these conditions in adult mice, suggesting that changes in other factors, such as osteoprotegerin or estrogen levels, play a dominant role.

MIP/MTMR14 and muscle aging

It is well known that the human aging process is associated with a progressive loss of muscular strength. Characteristic of this decline in muscle performance is the loss of skeletal muscle mass (sarcopenia) that occurs even in the healthy elderly. Indeed, humans can lose as much as 40% of their muscle mass from age 20 to 60. This significant loss of muscle mass and strength has important health and social implications as muscle weakness can contribute to an increased risk of falls and a loss of independence. Therefore, improving our understanding of the mechanisms responsible for sarcopenia and the age-related loss of muscle strength is important. The report by Romero-Suarez et al. [1] provides important new information regarding a potential cellular mechanism that may contribute to sarcopenia. These investigators used a combination of experimental approaches to phenotypically compare wild type old mice with mature mice lacking a novel muscle specific inositide phosphatase (MIP/MTMR14) that plays an important role in muscle calcium homeostasis. Interestingly, the mature MIP/MTMR14 knockout mice displayed phenotypes that closely resembled the muscles of old animals. Indeed, these relatively young knockout mice displayed impaired muscle calcium homeostasis, depressed muscle contractile force production and a loss of muscle mass that is commonly observed in senescent animals. Interestingly, the old wild type mice also displayed impaired muscle calcium homeostasis and decreases in muscle size and contractile function. Importantly, these old mice also possessed reduced muscle levels of MIP/MTMR14. The authors concluded that these findings were consistent with the hypothesis that an age-related loss in MIP/MTMR14 may be a contributory factor to sarcopenia. The findings of Romero-Suarez et al. [1] are exciting and set the stage for future experiments to further examine the molecular role that MIP/MTMR14 plays in the age-related decline in skeletal muscle mass and function. Prior studies by Shen, et al. [2] suggest that MIP/MTMR14 deficiency elevates PtdIns(3,4)P2 and PtdIns(3,5)P2 levels in the sarcoplasmic reticulum. These phosphoinositide derivatives appear to promote calcium leak, contractile dysfunction, and protein catabolism. It remains to be seen whether PtdIns(3,4)P2 and PtdIns(3,5)P2 accumulate in the SR of aged muscles. This is an intriguing possibility. A detailed understanding of the cellular function of MIP/MTMR14 and the molecular events leading to saropenia may lead to clinical countermeasures to slow the age-related loss of muscular strength.

A Candidate Gene for a Biological Marker of Schizophrenia in Mice

A Candidate Gene for a Biological Marker of Schizophrenia in Mice

RECS1 is a Negative Regulator of Matrix Metalloproteinase-9 Production and Aged RECS1 Knockout Mice are Prone to Aortic Dilation

RECS1 is a mechanical stress responsive gene and RECS1 knockout (KO) mice (older than 14 months) are prone to cystic medial degeneration (CMD). The present study was designed to assess whether RECS1 KO mice have altered gelatinase (matrix metalloproteinase (MMP)-2 and MMP-9) levels and whether they are prone to aortic dilation. Aortic and plasma gelatinase levels in RECS1 KO and wild-type (WT) mice were assessed by gelatin zymography and Western blot analysis. Pro-MMP-9 (in the aorta), neutrophil gelatinase-associated lipocalin/MMP-9 complex (NGAL-MMP-9, in plasma), and active-MMP-9 protein levels were more abundant in KO mice throughout adulthood compared with WT mice. Aortic MMP-2, aortic MMP-9, and plasma MMP-9 activation increased with age, even though the aortic pro-MMP-9, plasma NGAL-MMP-9, aortic and plasma pro-MMP-2 production decreased: this was true both for the WT and KO animals. Aortic pro-MMP-2, aortic active-MMP-2, and plasma pro-MMP-2 protein levels were higher in the aged KO mice, but they were lower in the young KO mice than those in WT mice. Thoracic aortic dilation was observed only in the aged KO mice. In situ zymographic experiments confirmed that the increased aortic gelatinase activities were associated with CMD and aortic dilation observed in the aged KO mice. RECS1 negatively regulates aortic MMP-9 production and knocking out RECS1 induces susceptibility to aortic dilation as well as CMD in the aged mice. The present study suggests that RECS1 plays protective roles in vascular remodeling. We speculate that inhibiting unfavorable deposition and extracellular matrix degradation are both important for prevention and treatment of aneurysms.

Fragile X mental retardation protein is necessary for neurotransmitter-activated protein translation at synapses.

Fragile X mental retardation is caused by absence of the RNA-binding protein fragile X mental retardation protein (FMRP), encoded by the FMR1 gene. There is increasing evidence that FMRP regulates transport and modulates translation of some mRNAs. We studied neurotransmitter-activated synaptic protein synthesis in fmr1-knockout mice. Synaptoneurosomes from knockout mice did not manifest accelerated polyribosome assembly or protein synthesis as it occurs in wild-type mice upon stimulation of group I metabotropic glutamate receptors. Direct activation of protein kinase C did not compensate in the knockout mouse, indicating that the FMRP-dependent step is further along the signaling pathway. Visual cortices of young knockout mice exhibited a lower proportion of dendritic spine synapses containing polyribosomes than did the cortices of wild-type mice, corroborating this finding in vivo. This deficit in rapid neurotransmitter-controlled local translation of specific proteins may contribute to morphological and functional abnormalities observed in patients with fragile X syndrome.

The liver in transforming growth factor-Beta-1 (TGF-beta 1) null mutant mice.

Transforming growth factor-beta-1 (TGF-beta 1) null mutant mice have no gross developmental abnormalities at birth but succumb to multifocal inflammatory lesions that lead to organ failure and death about 20 days after birth. Treatment with anti-inflammatory and immune suppressive agents, such as rapamycin, reduces the severity and extent of inflammatory infiltrates in the liver and can prolong the life of knockout (KO) mice compared to untreated null mice. To determine whether there is an associated hepatic phenotype, livers of "young" (< 3 weeks), "old" (> 3 weeks), and age-matched wild-type (WT) mice were studied using light and electronmicroscopy. On light microscopy, old KO mice had foci of mononuclear cells in liver parenchyma in addition to scattered foci of megalocytosis. Intracytoplasmic vacuoles, some of which were juxtanuclear in location, were also seen but these were most prominent in the oldest (10 weeks) rapamycin-treated mouse. In the untreated young KO mice, there were only foci of mononuclear cells in the liver parenchyma and portal tracts and variable numbers of binucleated hepatocytes. Ultrastructurally, there was a significant increase in the number of mitochondria in livers of the old KO mice, when compared either to the age-matched wild-type or to the young KO mice (p > .001). Hepatocytes from all KO mice showed increased numbers of hypertrophied or enlarged Golgi complexes compared to age-matched wild-type mice. Intracytoplasmic canaliculi lined with microvilli were seen in livers of old KO mice, but were absent in the young KO and wild-type mice. Primary cultures of hepatocytes, derived from livers of both young and old KO mice, showed similar changes on phase contrast and electronmicroscopy. These included juxtanuclear vacuoles, intracytoplasmic canaliculi, enlarged Golgi vesicles, and increased numbers of autolysosomes. Phenotypic abnormalities of mitochondria were either minimal or absent in cultured KO hepatocytes. The findings demonstrate, for the first time, that targeted disruption of the TGF-beta 1 gene in mice results in an altered ultrastructural phenotype of hepatocytes. The data suggest that TGF-beta 1 may be required for normal development and regulation of subcellular organelles in hepatocytes and may be essential for physiological functions involving mitochondria and Golgi complex.