Experimentally Induced Sepsis Causes Extensive Hypomyelination in the Prefrontal Cortex and Hippocampus in Neonatal Rats.

Neonatal sepsis is associated with cognitive deficit in the later life. Axonal myelination plays a pivotal role in neurotransmission and formation of learning and memory. This study aimed to explore if systemic lipopolysaccharide (LPS) injection would induce hypomyelination in the prefrontal cortex and hippocampus in developing septic neonatal rats. Sprague-Dawley rats (1-day old) were injected with LPS (1 mg/kg) intraperitoneally. By electron microscopy, axonal hypomyelination was evident in the subcortical white matter and hippocampus. The expression of myelin proteins including CNPase, MBP, PLP and MAG was downregulated in both areas of the brain at 7, 14 and 28 days after LPS injection. The frequency of MBP and PLP-positive oligodendrocyte was significantly reduced using in situ hybridization in the cerebral cortex and hippocampus at the corresponding time points after LPS injection, whereas the expression of NG2 and PDGFRα was noticeably increased. In tandem with this was reduction of Olig1 and Olig2 expressions which are involved in differentiation/maturation of OPCs. Expression of NFL, NFM, and NFH was significantly downregulated, indicating that axon development was disrupted after LPS injection. Morris Water Maze behavioral test, Open field test, Rotarod test, and Pole test were used to evaluate neurological behaviors of 28 days rats. The rats in the LPS group showed the impairment of motor coordination, balance, memory, and learning ability and represented bradykinesia and anxiety-like behavior. The present results suggest that following systemic LPS injection, differentiation/maturation of OPCs was affected which may be attributed to the inhibition of transcription factors Olig1 and Olig2 expression resulting in impairment to axonal development. It is suggested that this would ultimately lead to axonal hypomyelination in the prefrontal cortex and hippocampus, which may be associated with neurological deficits in later life.

Mechanical stretch induces myelin protein loss in oligodendrocytes by activating Erk1/2 in a calcium-dependent manner.

Myelin loss in the brain is a common occurrence in traumatic brain injury (TBI) that results from impact-induced acceleration forces to the head. Fast and abrupt head motions, either resulting from violent blows and/or jolts, cause rapid stretching of the brain tissue, and the long axons within the white matter tracts are especially vulnerable to such mechanical strain. Recent studies have shown that mechanotransduction plays an important role in regulating oligodendrocyte progenitors cell differentiation into oligodendrocytes. However, little is known about the impact of mechanical strain on mature oligodendrocytes and the stability of their associated myelin sheaths. We used an in vitro cellular stretch device to address these questions, as well as characterize a mechanotransduction mechanism that mediates oligodendrocyte responses. Mechanical stretch caused a transient and reversible myelin protein loss in oligodendrocytes. Cell death was not observed. Myelin protein loss was accompanied by an increase in intracellular Ca2+ and Erk1/2 activation. Chelating Ca2+ or inhibiting Erk1/2 activation was sufficient to block the stretch-induced loss of myelin protein. Further biochemical analyses revealed that the stretch-induced myelin protein loss was mediated by the release of Ca2+ from the endoplasmic reticulum (ER) and subsequent Ca2+ -dependent activation of Erk1/2. Altogether, our findings characterize an Erk1/2-dependent mechanotransduction mechanism in mature oligodendrocytes that de-stabilizes the myelination program.

Pain and small-fiber affection in hereditary neuropathy with liability to pressure palsies (HNPP).

Background and aims Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal - dominant hereditary neuropathy caused by a deficiency in the peripheral protein PMP-22, due to deletion on chromosome 17p11,2 or in some rare cases point mutations in the PMP-22 gene. The clinical picture is characterized by recurrent mononeuropathies in nerves which frequently may be exposed to pressure, such as the median, ulnar, radial and peroneal nerves or also a more general neuropathy. Although pain is reported to be an unusual clinical symptom, there have been reports of pain in a surprisingly high proportion of these patients. Since pain may be explained by mechanisms in afferent small unmyelinated C- nerve fibers, an assessment of the function of small nerve fibers has been requested. The purpose of the present study was to investigate the presence of pain and the possible affection of afferent small nerve-fibers, A-δ and C-fibers, by quantitative sensory testing (QST)-assessment of thermal thresholds, as well as quantitative sudomotor axon reflex (QSART), a quantitative, validated assessment of efferent postganglionic sumodotor function. QST values were compared to values of age- and sex matched healthy subjects. Methods The 19 patients were investigated clinically, with an emphasis on pain characteristics, with nerve conduction studies (NCS) of major nerves in upper- and lower extremity, small fiber testing (QST, measurement of thermal thresholds) and with QSART. Results A total of 10 patients reported numbness in some extremity, suggesting entrapment of individual nerves as well as a general neuropathy, as verified by NCS in nine patients. A total of 15 patients had findings compatible with a general polyneuropathy. A total of eight patients reported pain, seven patients with pain in the feet, described as burning, aching, shooting and six with severe pathological QST values, mainly cold detection, but also four patients with elevated thresholds to warmth. Four of the patients had signs of a severe sensory neuropathy on NCS, with no sural findings. One patient had only pain in the arms, with only minor changes on NCS and with normal QST-values. Cold detection thresholds (CD) were significantly elevated (reduced sensibility) on the dorsum of the foot (mean of two feet), in patients [26.0 °C (19.7-28.0)] as compared with healthy subjects [28.6 °C (27.4-29.6) p = 0.000]. There were also significantly elevated warmth detection thresholds (WD) in feet in patients 39.5 °C (36.4-42.9) compared to healthy subjects [37.7 °C (36.1-39.4) p = 0.048]. However, there were no significant differences in QST values between patients with and without pain. Conclusions Of a total of 19 patients with verified HNPP, eight patients (42.1%) suffered from neuropathic pain, mainly in both feet. Implications Due to the high percentage of pain in HNPP, it is important not to disregard this diagnosis in a patient presenting with pain. Since there are no significant differences in QST values in patients with and without pain, routine QST studies in HNPP do not seem necessary.

Peripheral myelin protein 22 modulates store-operated calcium channel activity, providing insights into Charcot-Marie-Tooth disease etiology.

Charcot-Marie-Tooth (CMT) disease is a peripheral neuropathy associated with gene duplication and point mutations in the peripheral myelin protein 22 (PMP22) gene. However, the role of PMP22 in Schwann cell physiology and the mechanisms by which PMP22 mutations cause CMT are not well-understood. On the basis of homology between PMP22 and proteins associated with modulation of ion channels, we hypothesized that PMP22 alters ion channel activity. Using whole-cell electrophysiology, we show here that heterologous PMP22 expression increases the amplitude of currents similar to those ascribed to store-operated calcium (SOC) channels, particularly those involving transient receptor canonical channel 1 (TrpC1). These channels help replenish Ca2+ in the endoplasmic reticulum (ER) following stimulus-induced depletion. Currents with similar properties were recorded in WT but not pmp22-/- mouse Schwann cells. Heterologous expression of the CMT-associated PMP22_L16P variant, which fails to reach the plasma membrane and localizes to the ER, led to larger currents than WT PMP22. Similarly, Schwann cells isolated from Trembler J (TrJ; PMP22_L16P) mice had larger currents than WT littermates. Calcium imaging in live nerves and cultured Schwann cells revealed elevated intracellular Ca2+ in TrJ mice compared with WT. Moreover, we found that PMP22 co-immunoprecipitated with stromal interaction molecule 1 (STIM1), the Ca2+ sensor SOC channel subunit in the ER. These results suggest that in the ER, PMP22 interacts with STIM1 and increases Ca2+ influx through SOC channels. Excess or mutant PMP22 in the ER may elevate intracellular Ca2+ levels, which could contribute to CMT pathology.

Terahertz Spectroscopic Diagnosis of Myelin Deficit Brain in Mice and Rhesus Monkey with Chemometric Techniques.

While myelin deficit of the central nervous system leads to several severe diseases, the definitive diagnostic means are lacking. We proposed and performed terahertz time-domain spectroscopy (THz-TDS) combined with chemometric techniques to discriminate and evaluate the severity of myelin deficit in mouse and rhesus monkey brains. The THz refractive index and absorption coefficient of paraffin-embedded brain tissues from both normal and mutant dysmyelinating mice are shown. Principal component analysis of time-domain THz signal (PCA-tdTHz) and absorption-refractive index relation of THz spectrum identified myelin deficit without exogenous labeling or any pretreatment. Further, with the established PCA-tdTHz, we evaluated the severity of myelin deficit lesions in rhesus monkey brain induced by experimental autoimmune encephalomyelitis, which is the most-studied animal model of multiple sclerosis. The results well matched the pathological analysis, indicating that PCA-tdTHz is a quick, powerful, evolving tool for identification and evaluation myelin deficit in preclinical animals and potentially in para-clinical human biopsy.

Mammalian-Specific Central Myelin Protein Opalin Is Redundant for Normal Myelination: Structural and Behavioral Assessments.

Opalin, a central nervous system-specific myelin protein phylogenetically unique to mammals, has been suggested to play a role in mammalian-specific myelin. To elucidate the role of Opalin in mammalian myelin, we disrupted the Opalin gene in mice and analyzed the impacts on myelination and behavior. Opalin-knockout (Opalin-/-) mice were born at a Mendelian ratio and had a normal body shape and weight. Interestingly, Opalin-/- mice had no obvious abnormalities in major myelin protein compositions, expression of oligodendrocyte lineage markers, or domain organization of myelinated axons compared with WT mice (Opalin+/+) mice. Electron microscopic observation of the optic nerves did not reveal obvious differences between Opalin+/+ and Opalin-/- mice in terms of fine structures of paranodal loops, transverse bands, and multi-lamellae of myelinated axons. Moreover, sensory reflex, circadian rhythm, and locomotor activity in the home cage, as well as depression-like behavior, in the Opalin-/- mice were indistinguishable from the Opalin+/+ mice. Nevertheless, a subtle but significant impact on exploratory activity became apparent in Opalin-/- mice exposed to a novel environment. These results suggest that Opalin is not critical for central nervous system myelination or basic sensory and motor activities under conventional breeding conditions, although it might be required for fine-tuning of exploratory behavior.

Nogo-A deletion increases the plasticity of the optokinetic response and changes retinal projection organization in the adult mouse visual system.

The inhibitory action of Nogo-A on axonal growth has been well described. However, much less is known about the effects that Nogo-A could exert on the plasticity of neuronal circuits under physiological conditions. We investigated the effects of Nogo-A knock-out (KO) on visual function of adult mice using the optokinetic response (OKR) and the monocular deprivation (MD)-induced OKR plasticity and analyzed the anatomical organization of the eye-specific retinal projections. The spatial frequency sensitivity was higher in intact Nogo-A KO than in wild-type (WT) mice. After MD, Nogo-A KO mice reached a significantly higher spatial frequency and contrast sensitivity. Bilateral ablation of the visual cortex did not affect the OKR sensitivity before MD but reduced the MD-induced enhancement of OKR by approximately 50% in Nogo-A KO and WT mice. These results suggest that cortical and subcortical brain structures contribute to the OKR plasticity. The tracing of retinal projections to the dorsal lateral geniculate nucleus (dLGN) revealed that the segregation of eye-specific terminals was decreased in the adult Nogo-A KO dLGN compared with WT mice. Strikingly, MD of the right eye led to additional desegregation of retinal projections in the left dLGN of Nogo-A KO but not in WT mice. In particular, MD promoted ectopic varicosity formation in Nogo-A KO dLGN axons. The present data show that Nogo-A restricts visual experience-driven plasticity of the OKR and plays a role in the segregation and maintenance of retinal projections to the brain.

Comprehensive Corticospinal Labeling with mu-crystallin Transgene Reveals Axon Regeneration after Spinal Cord Trauma in ngr1-/- Mice.

Spinal cord injury interrupts descending motor tracts and creates persistent functional deficits due to the absence of spontaneous axon regeneration. Of descending pathways, the corticospinal tract (CST) is thought to be the most critical for voluntary function in primates. Even with multiple tracer injections and genetic tools, the CST is visualized to only a minor degree in experimental studies. Here, we identify and validate the mu-crystallin (crym) gene as a high-fidelity marker of the CST. In transgenic mice expressing green fluorescent protein (GFP) under crym regulatory elements (crym-GFP), comprehensive and near complete CST labeling is achieved throughout the spinal cord. Bilateral pyramidotomy eliminated the 17,000 GFP-positive CST axons that were reproducibly labeled in brainstem from the spinal cord. We show that CST tracing with crym-GFP is 10-fold more efficient than tracing with biotinylated dextran amine (BDA). Using crym-GFP, we reevaluated the CST in mice lacking nogo receptor 1 (NgR1), a protein implicated in limiting neural repair. The number and trajectory of CST axons in ngr1(-/-) mice without injury was indistinguishable from ngr1(+/+) mice. After dorsal hemisection in the midthoracic cord, CST axons did not significantly regenerate in ngr1(+/+) mice, but an average of 162 of the 6000 labeled thoracic CST axons (2.68%) regenerated >100 µm past the lesion site in crym-GFP ngr1(-/-) mice. Although traditional BDA tracing cannot reliably visualize regenerating ngr1(-/-) CST axons, their regenerative course is clear with crym-GFP. Therefore the crym-GFP transgenic mouse is a useful tool for studies of CST anatomy in experimental studies of motor pathways. SIGNIFICANCE STATEMENT: Axon regeneration fails in the adult CNS, resulting in permanent functional deficits. Traditionally, inefficient extrinsic tracers such a biotinylated dextran amine (BDA) are used to label regenerating fibers after therapeutic intervention. We introduce crym-green fluorescent protein (GFP) transgenic mice as a comprehensive and specific tool with which to study the primary descending motor tract, the corticospinal tract (CST). CST labeling with crym-GFP is 10 times more efficient compared with BDA. The enhanced sensitivity afforded by crym-GFP revealed significant CST regeneration in NgR1 knock-out mice. Therefore, crym-GFP can be used as a standardized tool for future CST spinal cord injury studies.

Effects of PTEN and Nogo Codeletion on Corticospinal Axon Sprouting and Regeneration in Mice.

Axons in the adult CNS have poor ability to grow after injury, impeding functional recovery in patients of spinal cord injury. This has been attributed to both a developmental decline in neuron-intrinsic growth ability and the presence of extrinsic growth inhibitors. We previously showed that genetic deletion of Nogo, an extrinsic inhibitor, promoted axonal sprouting from uninjured corticospinal tract (CST) neurons but not regeneration from injured CST neurons, whereas genetic deletion of PTEN, an intrinsic inhibitor, promoted both CST sprouting and regeneration. Here we test the hypothesis that combining an elevation of neuron-intrinsic growth ability and a reduction of extrinsic growth inhibition by genetic codeletion of PTEN and Nogo may further improve injury-induced axonal growth. In an apparent paradox, additionally deleting Nogo further enhanced CST regeneration but not sprouting in PTEN-deleted mice. Enhanced CST regeneration and sprouting in PTEN and PTEN/Nogo-deleted mice were associated with no or only temporary improvement in functional recovery. Our data illustrate that neuron-intrinsic and -extrinsic factors regulate axon regeneration and sprouting in complex ways and provide proof-of-principle evidence that targeting both can further improve regeneration. Neuron-intrinsic growth ability is an important determinant of neuronal responsiveness to changes in extrinsic growth inhibition, such that an elevated intrinsic growth state is a prerequisite for reducing extrinsic inhibition to take effect on CST regeneration. Meanwhile, additional strategies are required to unleash the full potential for functional recovery with enhanced axon regeneration and/or sprouting.

Plasticity of intact rubral projections mediates spontaneous recovery of function after corticospinal tract injury.

Axons in the adult CNS fail to regenerate after injury, and therefore recovery from spinal cord injury (SCI) is limited. Although full recovery is rare, a modest degree of spontaneous recovery is observed consistently in a broad range of clinical and nonclinical situations. To define the mechanisms mediating spontaneous recovery of function after incomplete SCI, we created bilaterally complete medullary corticospinal tract lesions in adult mice, eliminating a crucial pathway for voluntary skilled movement. Anatomic and pharmacogenetic tools were used to identify the pathways driving spontaneous functional recovery in wild-type and plasticity-sensitized mice lacking Nogo receptor 1. We found that plasticity-sensitized mice recovered 50% of normal skilled locomotor function within 5 weeks of lesion. This significant, yet incomplete, spontaneous recovery was accompanied by extensive sprouting of intact rubrofugal and rubrospinal projections with the emergence of a de novo circuit between the red nucleus and the nucleus raphe magnus. Transient silencing of this rubro-raphe circuit in vivo via activation of the inhibitory DREADD (designer receptor exclusively activated by designer drugs) receptor hM4di abrogated spontaneous functional recovery. These data highlight the pivotal role of uninjured motor circuit plasticity in supporting functional recovery after trauma, and support a focus of experimental strategies on enhancing intact circuit rearrangement to promote functional recovery after SCI.

Reticulon 4 is necessary for endoplasmic reticulum tubulation, STIM1-Orai1 coupling, and store-operated calcium entry.

Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of RTN4, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling because of loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4-depleted cells fail to sustain elevated cytoplasmic Ca(2+) levels via SOCE and therefor are less susceptible to Ca(2+) overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4 in cellular Ca(2+) homeostasis.

NogoR1 and PirB signaling stimulates neural stem cell survival and proliferation.

Neural stem cells (NSCs) and neural progenitors (NPs) in the mammalian neocortex give rise to the main cell types of the nervous system. The biological behavior of these NSCs and NPs is regulated by extracellular niche derived autocrine-paracrine signaling factors on a developmental timeline. Our previous reports [Plos One 2010;5:e15341; J Neurochem 2011;117:565-578] have shown that chondroitin sulfate proteoglycan and ApolipoproteinE are autocrine-paracrine survival factors for NSCs. NogoA, a myelin related protein, is expressed in the cortical ventricular zones where NSCs reside. However, the functional role of Nogo signaling proteins in NSC behavior is not completely understood. In this study, we show that NogoA receptors, NogoR1 and PirB, are expressed in the ventricular zone where NSCs reside between E10.5 and 14.5 but not at E15.5. Nogo ligands stimulate NSC survival and proliferation in a dosage-dependent manner in vitro. NogoR1 and PirB are low and high affinity Nogo receptors, respectively and are responsible for the effects of Nogo ligands on NSC behavior. Inhibition of autocrine-paracrine Nogo signaling blocks NSC survival and proliferation. In NSCs, NogoR1 functions through Rho whereas PirB uses Shp1/2 signaling pathways to control NSC behavior. Taken together, this work suggests that Nogo signaling is an important pathway for survival of NSCs.

The sphingolipid receptor S1PR2 is a receptor for Nogo-a repressing synaptic plasticity.

Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) sphingosine 1-phosphate receptor 2 (S1PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S1PR2 on sites distinct from the pocket of the sphingolipid sphingosine 1-phosphate (S1P) and signals via the G protein G13, the Rho GEF LARG, and RhoA. Deleting or blocking S1PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S1PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A(-/-) mice, indicating a repressor function of the Nogo-A/S1PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S1PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S1PR2 signaling platforms will provide new insights into regulation of synaptic plasticity.

Knockdown of Peripheral Myelin Protein 22 Inhibits the Progression of Chronic Myeloid Leukemia.

We aimed to explore the underlying mechanism of peripheral myelin protein 22 (PMP22) in the development of chronic myeloid leukemia (CML). The level of PMP22 expression in CD34(+) cells isolated from CML patients' bone marrow samples (BMMCs) and peripheral blood samples (PBMCs) was determined by RT-PCR. In addition, PMP22-siRNA and scrambled control siRNA were transfected into human CML cell line K562 with Lipofectamine 2000 reagent. Cell viability and apoptosis were, respectively, determined by MTT assay and flow cytometry. Besides, the level of caspase 3 and Bcl-xL was then detected using Western blot. The level of PMP22 expression in CML patients' CD34(+) cells isolated from both PBMCs and BMMCs was significantly higher than the control group. PMP22 expression in K562 cells was successfully knocked down by siRNA. MTT analysis showed that knockdown of PMP22 inhibited the proliferation of CML cells. Flow cytometry showed that knockdown of PMP22 promoted the apoptosis of CML cells. Besides, Bcl-xL expression markedly decreased, while the expression of caspase 3 in CML cells significantly increased after knockdown of PMP22 expression. Our findings indicate that high expression of PMP22 may promote cell proliferation and inhibit cell apoptosis via upregulation of Bcl-xL or inhibition of caspase 3 activation, and thus may contribute to the development of CML. PMP22 may serve as a novel therapeutic target for the treatment of CML.

Abnormal junctions and permeability of myelin in PMP22-deficient nerves.

OBJECTIVE: The peripheral myelin protein-22 (PMP22) gene is associated with the most common types of inherited neuropathies, including hereditary neuropathy with liability to pressure palsies (HNPP) caused by PMP22 deficiency. However, the function of PMP22 has yet to be defined. Our previous study has shown that PMP22 deficiency causes an impaired propagation of nerve action potentials in the absence of demyelination. In the present study, we tested an alternative mechanism relating to myelin permeability. METHODS: Utilizing Pmp22(+) (/) (-) mice as a model of HNPP, we evaluated myelin junctions and their permeability using morphological, electrophysiological, and biochemical approaches. RESULTS: We show disruption of multiple types of cell junction complexes in peripheral nerve, resulting in increased permeability of myelin and impaired action potential propagation. We further demonstrate that PMP22 interacts with immunoglobulin domain-containing proteins known to regulate tight/adherens junctions and/or transmembrane adhesions, including junctional adhesion molecule-C (JAM-C) and myelin-associated glycoprotein (MAG). Deletion of Jam-c or Mag in mice recapitulates pathology in HNPP. INTERPRETATION: Our study reveals a novel mechanism by which PMP22 deficiency affects nerve conduction not through removal of myelin, but through disruption of myelin junctions.

Absence of Nogo-B (reticulon 4B) facilitates hepatic stellate cell apoptosis and diminishes hepatic fibrosis in mice.

Nogo-B (reticulon 4B) accentuates hepatic fibrosis and cirrhosis, but the mechanism remains unclear. The aim of this study was to identify the role of Nogo-B in hepatic stellate cell (HSC) apoptosis in cirrhotic livers. Cirrhosis was generated by carbon tetrachloride inhalation in wild-type (WT) and Nogo-A/B knockout (Nogo-B KO) mice. HSCs were isolated from WT and Nogo-B KO mice and cultured for activation and transformation to myofibroblasts (MF-HSCs). Human hepatic stellate cells (LX2 cells) were used to assess apoptotic responses of activated HSCs after silencing or overexpressing Nogo-B. Livers from cirrhotic Nogo-B KO mice showed significantly reduced fibrosis (P < 0.05) compared with WT mice. Apoptotic cells were more prominent in fibrotic areas of cirrhotic Nogo-B KO livers. Nogo-B KO MF-HSCs showed significantly increased levels of apoptotic markers, cleaved poly (ADP-ribose) polymerase, and caspase-3 and -8 (P < 0.05) compared with WT MF-HSCs in response to staurosporine. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, increased cleaved caspase-3 and -8 levels in Nogo-B KO MF-HSCs compared with WT MF-HSCs (P < 0.01). In LX2 cells, Nogo-B knockdown enhanced apoptosis in response to staurosporine, whereas Nogo-B overexpression inhibited apoptosis. The absence of Nogo-B enhances apoptosis of HSCs in experimental cirrhosis. Selective blockade of Nogo-B in HSCs may represent a potential therapeutic strategy to mitigate liver fibrosis.

Reticulon 4B (Nogo-B) facilitates hepatocyte proliferation and liver regeneration in mice.

UNLABELLED: Nogo-B, also known as reticulon 4B, promotes liver fibrosis and cirrhosis by facilitating the transforming growth factor ß (TGF-ß) signaling pathway in activated hepatic stellate cells. The aim of this study was to determine the role of Nogo-B in hepatocyte proliferation and liver regeneration. Partial hepatectomy (PHx, 70% resection) was performed in male wild-type (WT) and Nogo-A/B knockout mice (referred to as Nogo-B KO mice). Remnant livers were isolated 2 hours, 5 hours, and 1, 2, 3, 7, and 14 days after PHx. Hepatocyte proliferation was assessed by Ki67 labeling index. Quantitative real-time polymerase chain reaction was performed for genes known to be involved in liver regeneration. Hepatocytes isolated from WT and Nogo-B KO mice were used to examine the role of Nogo-B in interleukin-6 (IL-6), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and TGF-ß signaling. Nogo-B protein levels increased in the regenerating livers in a time-dependent manner after PHx. Specifically, Nogo-B expression in hepatocytes gradually spread from the periportal toward the central areas by 7 days after PHx, but receded notably by 14 days. Nogo-B facilitated IL-6/signal transducer and activator of transcription 3 signaling, increased HGF-induced but not EGF-induced hepatocyte proliferation, and tended to reduce TGF-ß1-induced suppression of hepatocyte proliferation in cultured hepatocytes. Lack of Nogo-B significantly induced TGF-ß1 and inhibitor of DNA binding expression 1 day after PHx and IL-6 and EGF expression 2 days after PHx. Lack of Nogo-B delayed hepatocyte proliferation but did not affect the liver-to-body ratio in the regenerative process. CONCLUSION: Nogo-B expression in hepatocytes facilitates hepatocyte proliferation and liver regeneration.

Neuronal Nogo-A negatively regulates dendritic morphology and synaptic transmission in the cerebellum.

Neuronal signal integration as well as synaptic transmission and plasticity highly depend on the morphology of dendrites and their spines. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. Nogo-A is also expressed by certain neurons, in particular during development, but its physiological function in this cell type is less well understood. We addressed this question in the cerebellum, where Nogo-A is transitorily highly expressed in the Purkinje cells (PCs) during early postnatal development. We used general genetic ablation (KO) as well as selective overexpression of Nogo-A in PCs to analyze its effect on dendritogenesis and on the formation of their main input synapses from parallel (PFs) and climbing fibers (CFs). PC dendritic trees were larger and more complex in Nogo-A KO mice and smaller than in wild-type in Nogo-A overexpressing PCs. Nogo-A KO resulted in premature soma-to-dendrite translocation of CFs and an enlargement of the CF territory in the molecular layer during development. Although spine density was not influenced by Nogo-A, the size of postsynaptic densities of PF-PC synapses was negatively correlated with the Nogo-A expression level. Electrophysiological studies revealed that Nogo-A negatively regulates the strength of synaptic transmission at the PF-PC synapse. Thus, Nogo-A appears as a negative regulator of PC input synapses, which orchestrates cerebellar connectivity through regulation of synapse morphology and the size of the PC dendritic tree.

Critical period for acoustic preference in mice.

Preference behaviors are often established during early life, but the underlying neural circuit mechanisms remain unknown. Adapting a unique nesting behavior assay, we confirmed a "critical period" for developing music preference in C57BL/6 mice. Early music exposure between postnatal days 15 and 24 reversed their innate bias for silent shelter, which typically could not be altered in adulthood. Instead, exposing adult mice treated acutely with valproic acid or carrying a targeted deletion of the Nogo receptor (NgR(-/-)) unmasked a strong plasticity of preference consistent with a reopening of the critical period as seen in other systems. Imaging of cFos expression revealed a prominent neuronal activation in response to the exposed music in the prelimbic and infralimbic medial prefrontal cortex only under conditions of open plasticity. Neither behavioral changes nor selective medial prefrontal cortex activation was observed in response to pure tone exposure, indicating a music-specific effect. Open-field center crossings were increased concomitant with shifts in music preference, suggesting a potential anxiolytic effect. Thus, music may offer both a unique window into the emotional state of mice and a potentially efficient assay for molecular "brakes" on critical period plasticity common to sensory and higher order brain areas.

A novel system to accelerate the progression of nerve degeneration in transgenic mouse models of neuropathies.

Axon degeneration is a common hallmark of many neurodegenerative diseases. There is now an abundance of spontaneous and genetically engineered mice available to study the mechanisms of axonal degeneration and to screen for axonal protective agents. However, many of these mouse models exhibit slow progressive axonal loss which can span over many months. Consequently, there is a pressing need to accelerate the pace of axonal loss over a short interval for high-throughput screening of pharmacological and genetic therapies. Here, we present a novel technique using acrylamide, an axonal neurotoxin, to provoke rapid axonal degeneration in murine models of neuropathies. The progressive axonal loss which typically occurs over 8 months was reproduced within 7 to 10 days of the acrylamide intoxication. This approach was successfully applied to Myelin Associated Glycoprotein knockout (MAG-/-) mouse and Trembler-J mouse, a popular murine model of Charcot-Marie-Tooth disease type 1 (CMT-1). Acrylamide intoxication in transgenic mouse models offers a novel experimental approach to accelerate the rate of axonal loss over short intervals for timely in vivo studies of nerve degeneration. This report also provides for the first time an animal model for medication or toxin-induced exacerbation of pre-existing neuropathies, a phenomenon widely reported in patients with neuropathies.