The LATS2 tumor suppressor inhibits SREBP and suppresses hepatic cholesterol accumulation.

The Hippo signaling pathway is a major regulator of organ size. In the liver, Hippo pathway deregulation promotes hyperplasia and hepatocellular carcinoma primarily through hyperactivation of its downstream effector, YAP. The LATS2 tumor suppressor is a core member of the Hippo pathway. A screen for LATS2-interacting proteins in liver-derived cells identified the transcription factor SREBP2, master regulator of cholesterol homeostasis. LATS2 down-regulation caused SREBP activation and accumulation of excessive cholesterol. Likewise, mice harboring liver-specific Lats2 conditional knockout (Lats2-CKO) displayed constitutive SREBP activation and overexpressed SREBP target genes and developed spontaneous fatty liver disease. Interestingly, the impact of LATS2 depletion on SREBP-mediated transcription was clearly distinct from that of YAP overexpression. When challenged with excess dietary cholesterol, Lats2-CKO mice manifested more severe liver damage than wild-type mice. Surprisingly, apoptosis, inflammation, and fibrosis were actually attenuated relative to wild-type mice, in association with impaired p53 activation. Subsequently, Lats2-CKO mice failed to recover effectively from cholesterol-induced damage upon return to a normal diet. Additionally, decreased LATS2 mRNA in association with increased SREBP target gene expression was observed in a subset of human nonalcoholic fatty liver disease cases. Together, these findings further highlight the tight links between tumor suppressors and metabolic homeostasis.

Cerebellar Learning Properties Are Modulated by the CRF Receptor.

Corticotropin-releasing factor (CRF) and its type 1 receptor (CRFR1) play an important role in the responses to stressful challenges. Despite the well established expression of CRFR1 in granular cells (GrCs), its role in procedural motor performance and memory formation remains elusive. To investigate the role of CRFR1 expression in cerebellar GrCs, we used a mouse model depleted of CRFR1 in these cells. We detected changes in the cellular learning mechanisms in GrCs depleted of CRFR1 in that they showed changes in intrinsic excitability and long-term synaptic plasticity. Analysis of cerebella transcriptome obtained from KO and control mice detected prominent alterations in the expression of calcium signaling pathways components. Moreover, male mice depleted of CRFR1 specifically in GrCs showed accelerated Pavlovian associative eye-blink conditioning, but no differences in baseline motor performance, locomotion, or fear and anxiety-related behaviors. Our findings shed light on the interplay between stress-related central mechanisms and cerebellar motor conditioning, highlighting the role of the CRF system in regulating particular forms of cerebellar learning.SIGNIFICANCE STATEMENT Although it is known that the corticotropin-releasing factor type 1 receptor (CRFR1) is highly expressed in the cerebellum, little attention has been given to its role in cerebellar functions in the behaving animal. Moreover, most of the attention was directed at the effect of CRF on Purkinje cells at the cellular level and, to this date, almost no data exist on the role of this stress-related receptor in other cerebellar structures. Here, we explored the behavioral and cellular effect of granular cell-specific ablation of CRFR1 We found a profound effect on learning both at the cellular and behavioral levels without an effect on baseline motor skills.

MRS of brain metabolite levels demonstrates the ability of scavenging of excess brain glutamate to protect against nerve agent induced seizures.

This study describes the use of in vivo magnetic resonance spectrocopy (MRS) to monitor brain glutamate and lactate levels in a paraoxon (PO) intoxication model. Our results show that the administration of recombinant glutamate-oxaloacetate transaminase (rGOT) in combination with oxaloacetate (OxAc) significantly reduces the brain-accumulated levels of glutamate. Previously we have shown that the treatment causes a rapid decrease of blood glutamate levels and creates a gradient between the brain and blood glutamate levels which leads to the efflux of excess brain glutamate into the blood stream thereby reducing its potential to cause neurological damage. The fact that this treatment significantly decreased the brain glutamate and lactate levels following PO intoxication suggests that it could become a new effective neuroprotective agent.

Store-operated Ca2+ entry regulatory factor alters murine metabolic state in an age-dependent manner via hypothalamic pathways.

Store-operated calcium entry (SOCE) is a vital process aimed at refilling cellular internal Ca2+ stores and a primary cellular signaling driver for transcription factors' entry to the nucleus. SOCE-associated regulatory factor (SARAF)/TMEM66 is an endoplasmic reticulum (ER)-resident transmembrane protein that promotes SOCE inactivation and prevents Ca2+ overfilling of the cell. Here, we demonstrate that mice deficient in SARAF develop age-dependent sarcopenic obesity with decreased energy expenditure, lean mass, and locomotion without affecting food consumption. Moreover, SARAF ablation reduces hippocampal proliferation, modulates the activity of the hypothalamus-pituitary-adrenal (HPA) axis, and mediates changes in anxiety-related behaviors. Interestingly, selective SARAF ablation in the hypothalamus's paraventricular nucleus (PVN) neurons reduces old age-induced obesity and preserves locomotor activity, lean mass, and energy expenditure, suggesting a possible central control with a site-specific role for SARAF. At the cellular level, SARAF ablation in hepatocytes leads to elevated SOCE, elevated vasopressin-induced Ca2+ oscillations, and an increased mitochondrial spare respiratory capacity (SPC), thus providing insights into the cellular mechanisms that may affect the global phenotypes. These effects may be mediated via the liver X receptor (LXR) and IL-1 signaling metabolic regulators explicitly altered in SARAF ablated cells. In short, our work supports both central and peripheral roles of SARAF in regulating metabolic, behavioral, and cellular responses.

Encephalopathy caused by ablation of very long acyl chain ceramide synthesis may be largely due to reduced galactosylceramide levels.

Sphingolipids (SLs) act as signaling molecules and as structural components in both neuronal cells and myelin. We now characterize the biochemical, histological, and behavioral abnormalities in the brain of a mouse lacking very long acyl (C22-C24) chain SLs. This mouse, which is defective in the ability to synthesize C22-C24-SLs due to ablation of ceramide synthase 2, has reduced levels of galactosylceramide (GalCer), a major component of myelin, and in particular reduced levels of non-hydroxy-C22-C24-GalCer and 2-hydroxy-C22-C24- GalCer. Noteworthy brain lesions develop with a time course consistent with a vital role for C22-C24-GalCer in myelin stability. Myelin degeneration and detachment was observed as was abnormal motor behavior originating from a subcortical region. Additional abnormalities included bilateral and symmetrical vacuolization and gliosis in specific brain areas, which corresponded to some extent to the pattern of ceramide synthase 2 expression, with astrogliosis considerably more pronounced than microglial activation. Unexpectedly, unidentified storage materials were detected in lysosomes of astrocytes, reminiscent of the accumulation that occurs in lysosomal storage disorders. Together, our data demonstrate a key role in the brain for SLs containing very long acyl chains and in particular GalCer with a reduction in their levels leading to distinctive morphological abnormalities in defined brain regions.

Unique in utero identification of fetuses in multifetal mouse pregnancies by placental bidirectional arterial spin labeling MRI.

Noninvasive imaging is a critical part of the study of developing embryos/fetuses, particularly in the context of alterations of gene expression in genetically modified animals. However, in litter-bearing animals, such as mice, the inability to accurately identify individual embryo/fetus in utero is a major obstacle to longitudinal, noninvasive in vivo studies. Arterial spin labeling MRI was adopted here to determine the fetal order along the uterine horns in vivo, based on the specific pattern of dual arterial blood supply within the mouse uterine horns. Blood enters the mouse uterus cranially through the ovarian artery and caudally through the uterine artery. Saturation slices were alternately placed on the maternal heart or on the bifurcation point of the common iliac artery, thereby saturating either downward inflow via the ovarian arteries or upward inflow via the uterine arteries, respectively. Saturation maps provided a unique signature with highly significant correlation between the direction-dependent magnetization transfer and the position of the fetuses/placentas along the uterine horns. The bidirectional arterial spin labeling-MRI method reported here opens possibilities to determine and pursue phenotypic alterations in fetuses and placentas in longitudinal studies of transgenic and knockout mice models, and for studying defects in placental vascular architecture.

Prediction of Ovarian Follicular Dominance by MRI Phenotyping of Hormonally Induced Vascular Remodeling.

In the mammalian female, only a small subset of ovarian follicles, known as the dominant follicles (DFs), are selected for ovulation in each reproductive cycle, while the majority of the follicles and their resident oocytes are destined for elimination. This study aimed at characterizing early changes in blood vessel properties upon the establishment of dominance in the mouse ovary and application of this vascular phenotype for prediction of the follicles destined to ovulate. Sexually immature mice, hormonally treated for induction of ovulation, were imaged at three different stages by dynamic contrast-enhanced (DCE) MRI: prior to hormonal administration, at the time of DF selection, and upon formation of the corpus luteum (CL). Macromolecular biotin-bovine serum albumin conjugated with gadolinium-diethylenetriaminepentaacetic acid (b-BSA-GdDTPA) was intravenously injected, and the dynamics of its extravasation from permeable vessels as well as its accumulation in the antral cavity of the ovarian follicles was followed by consecutive T1-weighted MRI. Permeability surface area product (permeability) and fractional blood volume (blood volume) were calculated from b-BSA-GdDTPA accumulation. We found that the neo-vasculature during the time of DF selection was characterized by low blood volume and low permeability values as compared to unstimulated animals. Interestingly, while the vasculature of the CL showed higher blood volume compared to the DF, it exhibited a similar permeability. Taking advantage of immobilized ovarian imaging, we combined DCE-MRI and intravital light microscopy, to reveal the vascular properties of follicles destined for dominance from the non-ovulating subordinate follicles (SFs). Immediately after their selection, permeability of the vasculature of DF was attenuated compared to SF while the blood volume remained similar. Furthermore, DFs were characterized by delayed contrast enhancement in the avascular follicular antrum, reflecting interstitial convection, whereas SFs were not. In this study, we showed that although DF selection is accompanied by blood vessel growth, the new vasculature remained relatively impermeable compared to the vasculature in control animal and compared to SF. Additionally, DFs show late signal enhancement in their antrum. These two properties may aid in clinical prediction of follicular dominance at an early stage of development and help in their diagnosis for possible treatment of infertility.

Brain pathology and cerebellar purkinje cell loss in a mouse model of chronic neuronopathic Gaucher disease.

Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba-/-;Gbatg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba-/-;Gbatg mice display behavioral abnormalities at ∼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba-/-;Gbatg mice being a genuine type 3 GD model. Together, the Gba-/-;Gbatg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients.

Piezo2 expressed in proprioceptive neurons is essential for skeletal integrity.

In humans, mutations in the PIEZO2 gene, which encodes for a mechanosensitive ion channel, were found to result in skeletal abnormalities including scoliosis and hip dysplasia. Here, we show in mice that loss of Piezo2 expression in the proprioceptive system recapitulates several human skeletal abnormalities. While loss of Piezo2 in chondrogenic or osteogenic lineages does not lead to human-like skeletal abnormalities, its loss in proprioceptive neurons leads to spine malalignment and hip dysplasia. To validate the non-autonomous role of proprioception in hip joint morphogenesis, we studied this process in mice mutant for proprioceptive system regulators Runx3 or Egr3. Loss of Runx3 in the peripheral nervous system, but not in skeletal lineages, leads to similar joint abnormalities, as does Egr3 loss of function. These findings expand the range of known regulatory roles of the proprioception system on the skeleton and provide a central component of the underlying molecular mechanism, namely Piezo2.

Assessing Mucosal Inflammation in a DSS-Induced Colitis Mouse Model by MR Colonography.

Inflammatory bowel disease (IBD) is characterized by a chronic flaring inflammation of the gastrointestinal tract. To determine disease activity, the inflammatory state of the colon should be assessed. Endoscopy in patients with IBD aids visualization of mucosal inflammation. However, because the mucosa is fragile, there is a significant risk of perforation. In addition, the technique is based on grading of the entire colon, which is highly operator-dependent. An improved, noninvasive, objective magnetic resonance imaging (MRI) technique will effectively assess pathologies in the small intestinal mucosa, more specifically, along the colon, and the bowel wall and surrounding structures. Here, dextran sodium sulfate polymer induced acute colitis in mice that was subsequently characterized by multisection magnetic resonance colonography. This study aimed to develop a noninvasive, objective, quantitative MRI technique for detecting mucosal inflammation in a dextran sodium sulfate-induced colitis mouse model. MRI results were correlated with endoscopic and histopathological evaluations.

The Proprioceptive System Masterminds Spinal Alignment: Insight into the Mechanism of Scoliosis.

Maintaining posture requires tight regulation of the position and orientation of numerous spinal components. Yet, surprisingly little is known about this regulatory mechanism, whose failure may result in spinal deformity as in adolescent idiopathic scoliosis. Here, we use genetic mouse models to demonstrate the involvement of proprioception in regulating spine alignment. Null mutants for Runx3 transcription factor, which lack TrkC neurons connecting between proprioceptive mechanoreceptors and spinal cord, developed peripubertal scoliosis not preceded by vertebral dysplasia or muscle asymmetry. Deletion of Runx3 in the peripheral nervous system or specifically in peripheral sensory neurons, or of enhancer elements driving Runx3 expression in proprioceptive neurons, induced a similar phenotype. Egr3 knockout mice, lacking muscle spindles, but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types may be required for this regulatory mechanism. These findings uncover a central role for the proprioceptive system in maintaining spinal alignment.

Chronic cholinergic imbalances promote brain diffusion and transport abnormalities.

Cholinergic imbalances occur after traumatic effects and in the initial stages of neurodegenerative diseases, but their long-lasting effects remained largely unexplained. To address this, we used TgS transgenic mice constitutively overexpressing synaptic acetylcholinesterase (AChE-S) and presenting a complex phenotype of progressive neurodeterioration. T1- and T2-weighted magnetic resonance (MR) brain images appeared similar. However, diffusion-weighted MRI showed decreased baseline water apparent diffusion coefficient in the brains of TgS animals. Furthermore, contrast-enhanced MRI after gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) injection demonstrated slower recovery of normal signals in the TgS brains than with controls. Perfusion MR imaging and difference T1 maps calculated from pre- postcontrast T1-weighted MR images indicated accumulation of more Gd-DTPA molecules in the TgS brains than in the parent strain, reflecting impaired blood-brain barrier (BBB) functioning in these transgenic mice. To explore the molecular mechanism(s) underlying these global phenotypes, we performed microarray analysis in the stress-controlling prefrontal cortex of TgS vs. strain-matched wild-type animals. Profound overexpression of numerous ion channels, transporters, and adhesion genes was confirmed by real time RT-PCR tests. Immunohistochemical and immunoblot analyses revealed corresponding increases in the level and cellular distributions of the chloride channel CLCN3 and the water channel AQP4, both of which contribute to BBB maintenance. Our study attributes to balanced cholinergic neurotransmission, a central role in the brain's maintenance of water diffusion and ion transport, and indicates that chronic impairments in this maintenance facilitate neurodeterioration through interference with BBB function.

High b-value q-space diffusion MRI in myelin-deficient rat spinal cords.

In this study, we explore the effect of the lack of myelin on the diffusion characteristics and diffusion anisotropy obtained from high b-value q-space diffusion-weighted MRI (q-space DWI) in excised rat spinal cords. Twenty-one-day-old myelin-deficient (md) mutant (N=6) and control rats (N=6) were used in this study. The MRI protocol included multi-slice T(1), T(2), proton density (PD) MR images and high b-value q-space diffusion MRI measured perpendicular and parallel to the fibers of the spine. q-Space displacement and probability maps, in both directions, as well as displacement anisotropy maps, were computed from the diffusion data. At the end of the MRI protocol, representative spinal cords from both groups were subjected to electron microscopy (EM). The md spinal cords show different gray/white matter contrast in the T(1), T(2) and PD MR images as compared with controls. In addition, the mean displacement extracted from the high b-value q-space diffusion data was found to be dramatically higher in the white matter (WM) of the md spinal cords than the controls when diffusion was measured perpendicular and parallel to the fibers of the spine. However, interestingly, at the diffusion time used in the present study, the difference in the WM displacement anisotropies of the two groups was not found to be statistically significant. Myelin was found to have a pronounced effect on the diffusion characteristics of water in WM but less so on the diffusion anisotropy observed at the diffusion time used in the present study.

Structural changes in glutamate cell swelling followed by multiparametric q-space diffusion MR of excised rat spinal cord.

Diffusion in the extracellular and intracellular spaces (ECS and ICS, respectively) was evaluated in excised spinal cords, before and after cell swelling induced by glutamate, by high b-value q-space diffusion MR of specific markers and water. The signal decays of deuterated tetramethylammonium (TMA-d(12)) chloride, an exogenous marker of the ECS, and N-acetyl aspartate (NAA), an endogenous marker of the ICS, were found to be non-mono-exponential at all diffusion times. The signal decays of these markers were found to depend on the diffusion time and the cell swelling induced by the glutamate. It was found, for example, that the mean displacements of the apparent fast and slow diffusion components of TMA-d(12) are 7.21 +/- 0.11 and 1.16 +/- 0.05 microm, respectively at a diffusion time of 496 ms. After exposure of the spinal cords to 10 mM of glutamate, these values decreased to 6.62 +/- 0.13 and 1.01 +/- 0.05 microm, respectively. The mean displacement of NAA, however, showed a less pronounced opposite trend and increased after cell swelling induced by exposure to glutamate. q-Space diffusion MR of water was found to be sensitive to exposure to glutamate, and q-space diffusion MRI showed that a more pronounced decrease in the apparent diffusion coefficient and the mean displacement of water is observed in the gray matter (GM) of the spinal cord. All these changes demonstrate that diffusion MR is indeed sensitive to structural changes caused by cell swelling induced by glutamate. Multiparametric high b-value q-space diffusion MR is useful for obtaining microstructural information in neuronal tissues.

Rasagiline prevents neurodegeneration in thiamine deficient rats-a longitudinal MRI study.

Neuroprotection is a therapeutic approach for the management of neurodegenerative diseases. Experimental thiamine deficiency (TD) in rats provides a model for selective neurodegeneration accompanied by chronic oxidative deficits. Rats exhibit neurological and cognitive impairments, which can be partially reversed by thiamine administration, enabling the study of mechanisms of neurodegeneration as well as neuroprotection. In this magnetic resonance (MR) study we used various techniques to characterize the neuroprotective effects of rasagiline, a selective MAO-B inhibitor. TD was induced by a thiamine-deficient diet and daily injections of the central thiamine antagonist, pyrithiamine. Daily injections of either saline or rasagiline (3mg/kg) were also administered to untreated-TD rats and rasagiline-treated TD rats respectively. With the appearance of neurological symptoms, all injections were terminated and thiamine was restored. MRI scans were performed before induction of TD (control values), on days 10, 12 (before symptoms appear), 14 (symptomatic stage) and during the recuperation period. Both groups were assessed using in-vivo serial T2-weighted imaging and diffusion tensor imaging (DTI), from which apparent diffusion coefficient (ADC) and fractional anisotropy (FA) maps were calculated. A histopathological evaluation was correlated with the MRI analysis. Thalamic hyperintensities were significantly smaller and less severe in the rasagiline-treated TD rats. Enlargement of the lateral ventricles was significantly less pronounced in the rasagiline-treated TD group. FA values of the untreated-TD group decreased significantly in the thalamic on days 12 and 14 and in the corpus callosum on day 14. These results demonstrate significant neuroprotection by rasagiline which could have implications for clinical neurodegenerative disorders.

Neurodegeneration in thiamine deficient rats-A longitudinal MRI study.

Selective neurodegeneration accompanied by mitochondrial dysfunction characterizes neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Thiamine deficiency (TD) in rats is a model for the study of cellular and molecular mechanisms that lead to selective neuronal loss caused by chronic oxidative deficits. Neurodegeneration in TD-rats develops over a period of 12 to 14 days and can be partially reversed by thiamine administration. The aim of this study was to characterize the in-vivo progression of neurodegeneration and the neuronal rescue processes in TD using T(2) magnetic resonance mapping and diffusion tensor imaging (DTI). Each rat was scanned prior to TD induction (day 0), before the appearance of neurological symptoms (day 10), during the symptomatic stage (days 12 and 14) and during the recuperation period (days 31 and 87). Time-dependent lesions were revealed mainly in the thalamus and the inferior colliculi. Early decrease in the fractional anisotropy (FA) was found on day 10 in the inferior colliculi and to a lesser degree in the thalamus, while the earliest detectable changes in the T(2) parameter occurred only on day 12. FA values in the thalamus remained significantly low after thiamine restoration, suggesting irreversible disarrangement and replacement of neuronal structures. While T(2) values in the frontal cortex demonstrated no lesions, FA values significantly increased on days 14 and 31. An enlargement of the lateral ventricles was observed and persevered during the recovery period. This longitudinal MRI study demonstrated that in TD MRI can detect neurodegeneration and neuronal recovery. DTI is more sensitive than T(2) mapping in the early detection of TD lesions.

A novel Diclofenac-carrier for local treatment of osteoarthritis applying live-animal MRI.

The prevailing chronic treatment for osteoarthritis--oral administration of NSAIDs--is accompanied by severe adverse effects and risks of gastrointestinal (GI) toxicity. The working hypothesis of this study was that increased NSAID-efficacy and alleviation of adverse effects can be achieved by local administration of a new slow-release NSAID-carrier formulation. Diclofenac was the test NSAID and collagomers--novel vesicular-shaped microparticles based on collagen-lipid conjugates--were the carriers. Collagomers were stable in simulated synovial fluid and showed: high-efficiency drug encapsulation (85%); slow drug release (tau((1/2))=11 days); high affinity to target cells (Kd=2.6 nM collagen). In vitro activity of Diclofenac released from the carriers was similar to fresh drug solutions. Diclofenac-collagomer therapeutic effects were studied in osteoarthritis-induced rats, using live-animal MRI. A single intra-articular injection of the Diclofenac-collagomer formulation reduced inflammation over 3 weeks significantly vs. untreated animals (p<0.001), and vs. the conventional treatment which is free drug PerOs (p<0.03). Bypassing the GI, the novel treatment circumvents adverse effects of the conventional approach. In conclusion, the collagomers performed as functional Diclofenac-depots for local treatment of osteoarthritis, avoiding GI adverse effects. The in vivo results merit further investigations of this novel NSAID formulation as a valid option to the conventional treatment.

Improved detectability of experimental allergic encephalomyelitis in excised swine spinal cords by high b-value q-space DWI.

Experimental allergic encephalomyelitis (EAE) is the primary experimental model of multiple sclerosis (MS), which involves both inflammation and demyelination and is known to be species-dependent. Spinal cord abnormalities were found in more than 80% of postmortem specimens of MS patients. In the present study, T1, T2 and high b-value q-space diffusion-weighted magnetic resonance imaging (MRI) were used, for the first time, to characterize the EAE model in excised swine spinal cords. The MR images were compared with histological staining and clinical scoring. Although all spinal cords were excised from swine with severe or very severe (clinical score between 3 to 5 on a scale of 5) motor impairments, T1- and T2-weighted MRI revealed white matter (WM) abnormalities in only five of the ten EAE diseased spinal cords studied, while high b-value q-space diffusion weighted MRI (q-space DWI) detected WM abnormalities in all diseased spinal cords studied. Interestingly, high b-value q-space DWI was able to detect abnormalities in the normal appearing white matter (NAWM) even in spinal cords where no plaques were identified by the T1- and T2-weighted MR images. Good anatomical correlation was observed between the high b-value q-space MR images and histology. The extent of DWI abnormalities paralleled the clinical scoring and correlated with histology. In addition, areas classified as NAWM by the T1- and T2-weighted MR images that showed abnormalities in the q-space DWI were also found to have abnormal histology. This improved detection level of the EAE model by high b-value q-space DWI over conventional T1-, and T2-weighted MRI is briefly discussed.