Critical neuroprotective roles of heme oxygenase‐1 induction against axonal injury‐induced retinal ganglion cell death

Although axonal damage induces significant retinal ganglion cell (RGC) death, small numbers of RGCs are able to survive up to 7 days after optic nerve crush (NC) injury. To develop new treatments, we set out to identify patterns of change in the gene expression of axonal damage‐resistant RGCs. To compensate for the low density of RGCs in the retina, we performed retrograde labeling of these cells with 4Di‐10ASP in adult mice and 7 days after NC purified the RGCs with fluorescence‐activated cell sorting. Gene expression in the cells was determined with a microarray, and the expression of Ho‐1 was determined with quantitative PCR (qPCR). Changes in protein expression were assessed with immunohistochemistry and immunoblotting. Additionally, the density of Fluoro‐gold‐labeled RGCs was counted in retinas from mice pretreated with CoPP, a potent HO‐1 inducer. The microarray and qPCR analyses showed increased expression of Ho‐1 in the post‐NC RGCs. Immunohistochemistry also showed that HO‐1‐positive cells were present in the ganglion cell layer (GCL), and cell counting showed that the proportion of HO‐1‐positive cells in the GCL rose significantly after NC. Seven days after NC, the number of RGCs in the CoPP‐treated mice was significantly higher than in the control mice. Combined pretreatment with SnPP, an HO‐1 inhibitor, suppressed the neuroprotective effect of CoPP. These results reflect changes in HO‐1 activity to RGCs that are a key part of RGC survival. Upregulation of HO‐1 signaling may therefore be a novel therapeutic strategy for glaucoma. © 2014 Wiley Periodicals, Inc.     

Regression of retinal capillaries following N‐methyl‐D‐aspartate‐induced neurotoxicity in the neonatal rat retina

Degeneration of retinal capillaries occurs following N‐methyl‐D ‐aspartate (NMDA)‐induced retinal neurotoxicity, and the degree of capillary degeneration decreases in an age‐dependent manner. To determine the role of vascular endothelial growth factor (VEGF) in the high susceptibility of capillaries to neuronal damage during the early postnatal stage, this study compares the vascular regression patterns between NMDA‐treated retinas and retinas treated with N‐[2‐chloro‐4‐{(6,7‐dimethoxy‐4‐quinazolinyl)oxy}phenyl]‐N′‐propylurea (KRN633), a VEGF receptor tyrosine kinase inhibitor, in neonatal rats. Two days after a single intravitreal injection of NMDA (200 nmol/eye) on postnatal day (P) 7, substantial retinal neuron loss and delayed expansion of the retinal vascular bed were observed. The reduction in the capillary density in the central retina reached statistical significance 4 days after NMDA treatment. In retinas of rats injected subcutaneously with KRN633 (10 mg/kg) on P7 and P8, simplified vasculature attributable to capillary regression and prevention of endothelial cell growth were seen on P9, whereas no visible changes in the morphology of the retinal layers were observed. The degree of capillary degeneration in NMDA‐treated retinas was less than that in KRN633‐treated retinas. No apparent changes in immunoreactivities for VEGF were found 2 days after NMDA treatment. These results indicate that neuronal cell loss in the retina precedes retinal capillary degeneration following NMDA treatment, and VEGF‐dependent immature capillaries might be more susceptible to NMDA‐induced neuronal damage. © 2014 Wiley Periodicals, Inc.     

Virally delivered,constitutively active NFκB improves survival of injured retinal ganglion cells

As axon damage and retinal ganglion cell (RGC) loss lead to blindness, therapies that increase RGC survival and axon regrowth have direct clinical relevance. Given that NFκB signaling is critical for neuronal survival and may regulate neurite growth, we investigated the therapeutic potential of NFκB signaling in RGC survival and axon regeneration. Although both NFκB subunits (p65 and p50) are present in RGCs, p65 exists in an inactive (unphosphorylated) state when RGCs are subjected to neurotoxic conditions. In this study, we used a phosphomimetic approach to generate DNA coding for an activated (phosphorylated) p65 (p65mut), then employed an adeno‐associated virus serotype 2 (AAV2) to deliver the DNA into RGCs. We tested whether constitutive p65mut expression prevents death and facilitates neurite outgrowth in RGCs subjected to transient retinal ischemia or optic nerve crush (ONC), two models of neurotoxicity. Our data indicate that RGCs treated with AAV2‐p65mut displayed a significant increase in survival compared to controls in ONC model (77 ± 7% vs. 25 ± 3%, P‐value = 0.0001). We also found protective effect of modified p65 in RGCs of ischemic retinas (55 ± 12% vs. 35 ± 6%), but not to a statistically significant degree (P‐value = 0.14). We did not detect a difference in axon regeneration between experimental and control animals after ONC. These findings suggest that increased NFκB signaling in RGCs attenuates retinal damage in animal models of neurodegeneration, but insignificantly impacts axon regeneration.     

Dopamine modulates the off pathway in light‐adapted mouse retina

DL ‐2‐Amino‐4‐phosphonobutyric acid (APB) is often used as a tool to block On pathways in studies of interactions between On and Off pathways in retinas. APB is an agonist of mGluR6 receptors and hyperpolarizes the On cone bipolar cells and rod bipolar cells. How APB affects Off responses of retinal ganglion cells (RGCs) in mouse retinas under dark and light adaptation is not clear. The light‐evoked excitatory postsynaptic currents (light‐evoked EPSCs) from Off and On–Off RGCs cells were recorded using whole‐cell patch‐clamp recording to assess how APB affects Off responses (light‐evoked Off EPSCs) of RGCs in dark‐ and light‐adapted mouse retinas. We found that APB differentially affected Off responses of RGCs in dark‐ and light‐adapted mouse retinas. Under dark adaptation, while the APB‐sensitive Off responses were blocked, APB increased the remaining Off responses (mainly from the secondary rod Off pathways) via removal of inhibition from On pathways to Off pathways. Under light adaptation, APB decreased Off responses. Glycinergic and GABAergic antagonists did not prevent the APB‐induced reduction of Off responses of RGCs; however, a dopaminergic type 1 receptor (D₁) blocker (SCH 23390) and a hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channel blocker (ZD 7288) prevented the APB‐induced reduction of Off responses of RGCs under light adaptation. The results indicated afunctional circuit: On cone bipolar cells to Off cone bipolar cells via D₁ receptors and HCN channels. © 2012 Wiley Periodicals, Inc.     

Impact of melatonin receptor‐signaling on Zeitgeber time‐dependent changes in cell proliferation and apoptosis in the adult murine hippocampus

The hippocampus is subjected to diurnal/circadian rhythms on both the morphological and molecular levels. Certain aspects of cell proliferation in the adult hippocampus are regulated by melatonin and accompanied by apoptosis to ensure proper tissue maintenance and function. The present study investigated Zeitgeber time (ZT)‐dependent changes in cell proliferation and apoptosis in the adult murine hippocampus and their regulation by melatonin receptor type1 and type2 (MT1/2)‐mediated signaling. Adult melatonin‐proficient C3H/HeN mice and melatonin‐proficient (C3H/HeN) mice with targeted deletion of MT1/2 were adapted to a 12‐h light, 12‐h dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase‐3 in combination with different markers for the diverse cell types residing in the hippocampus served to identify and quantify proliferating and apoptotic cells in the hippocampal subregions. ZT‐dependent changes in cell proliferation and apoptosis were found exclusively in the subgranular zone (SGZ) and granule cell layer (GCL) of melatonin‐proficient mice with functional MT1/2. Cell proliferation in the SGZ showed ZT‐dependent changes indicated by an increase of proliferating immature neurons during the dark phase of the 24‐h light‐dark cycle. Apoptosis showed ZT‐dependent changes in the SGZ and GCL indicated by an increase of apoptotic immature neurons at ZT06 (SGZ) and a decrease of immature and mature neurons at ZT18 (GCL). Our results indicate that ZT‐dependent changes in proliferation of immature neurons in the SGZ are counterbalanced by ZT‐dependent changes in apoptosis of immature and mature neurons in the SGZ and GCL exclusively in mice with functional MT1/2. Therefore, MT1/2‐mediated signaling appears to be crucial for generation and timing of ZT‐dependent changes in cell proliferation and apoptosis and for differentiation of proliferating cells into neurons in the SGZ. © 2017 Wiley Periodicals, Inc.     

Genome‐wide gene expression profiling in GluR1 knockout mice: key role of the calcium signaling pathway in glutamatergically mediated hippocampal transmission

α‐Amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptors (AMPARs) convey fast synaptic transmission in the CNS and mediate various forms of hippocampal plasticity. Disruption of glutamate receptor type 1 (GluR1), a member of the AMPAR family, causes synaptic alterations and learning/memory deficits in mice. To gain mechanistic insight into the synaptic and behavioral changes associated with GluR1 deletion, hippocampal genome‐wide expression profiling was conducted using groups of GluR1 knockout (KO) mice and their wild‐type littermates. Regulation of 38 genes was found to be altered more than 30% (P < 0.01, n = 8), and seven of these genes were studied with additional quantitative experiments. A large portion of the altered genes encoded molecules involved in calcium signaling, including calcium channel components, calcium‐binding proteins and calcium‐calmodulin‐dependent protein kinase II subunits. At the protein level, we further evaluated some genes in the calcium pathway that were altered in GluR1 KO mice. Protein levels of two key molecules in the calcium pathway – GluR, ionotropic, N‐methyl‐d ‐aspartate‐1 and calcium/calmodulin‐dependent protein kinase II alpha – showed similar changes to those observed in mRNA levels. These findings raise the possibility that calcium signaling and other plasticity molecules may contribute to the hippocampal plasticity and behavioral deficits observed in GluR1 KO mice.     

Altered astrocyte morphology and vascular development in dystrophin‐Dp71‐null mice

Understanding retinal vascular development is crucial because many retinal vascular diseases such as diabetic retinopathy (in adults) or retinopathy of prematurity (in children) are among the leading causes of blindness. Given the localization of the protein Dp71 around the retinal vessels in adult mice and its role in maintaining retinal homeostasis, the aim of this study was to determine if Dp71 was involved in astrocyte and vascular development regulation. An experimental study in mouse retinas was conducted. Using a dual immunolabeling with antibodies to Dp71 and anti‐GFAP for astrocytes on retinal sections and isolated astrocytes, it was found that Dp71 was expressed in wild‐type (WT) mouse astrocytes from early developmental stages to adult stage. In Dp71‐null mice, a reduction in GFAP‐immunopositive astrocytes was observed as early as postnatal day 6 (P6) compared with WT mice. Using real‐time PCR, it was showed that Dp71 mRNA was stable between P1 and P6, in parallel with post‐natal vascular development. Regarding morphology in Dp71‐null and WT mice, a significant decrease in overall astrocyte process number in Dp71‐null retinas at P6 to adult age was found. Using fluorescence‐conjugated isolectin Griffonia simplicifolia on whole mount retinas, subsequent delay of developing vascular network at the same age in Dp71‐null mice was found. An evidence that the Dystrophin Dp71, a membrane‐associated cytoskeletal protein and one of the smaller Duchenne muscular dystrophy gene products, regulates astrocyte morphology and density and is associated with subsequent normal blood vessel development was provided. GLIA 2016;64:716–729     

Age‐dependent neuromuscular impairment in prion protein knockout mice

Introduction: The cellular prion protein (PrP^C) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP^C is still ambiguous. Evidence has suggested that PrP^C is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. Methods: We analyzed the age‐dependent influence of PrP^C on treadmill test–based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild‐type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP^C. Results: We found that aged PrP‐KO mice displayed a reduction in treadmill performance compared with PrP‐expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. Conclusion: PrP‐KO mice have an age‐dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations. Muscle Nerve 53: 269–279, 2016     

Essential role of endogenous calcitonin gene‐related peptide in pain‐associated plasticity in the central amygdala

The role of the neuropeptide calcitonin gene‐related peptide (CGRP) is well established in nociceptive behaviors. CGRP is highly expressed in the projection pathway from the parabrachial nucleus to the laterocapsular region of the central amygdala (CeC), which plays a critical role in relaying nociceptive information. The CeC is a key structure in pain behavior because it integrates and modulates nociceptive information along with other sensory signals. Previous studies have demonstrated that blockade of the amygdalar CGRP‐signaling cascade attenuates nociceptive behaviors in pain models, while CGRP application facilitates amygdalar synaptic transmission and induces pain behaviors. Despite these lines of evidence, it remains unclear whether endogenous CGRP is involved in the development of nociceptive behaviors accompanied with amygdalar plasticity in a peripheral inflammation model in vivo. To directly address this, we utilized a previously generated CGRP knockout (KO) mouse to longitudinally study formalin‐induced plasticity and nociceptive behavior. We found that synaptic potentiation in the right PB‐CeC pathway that was observed in wild‐type mice was drastically attenuated in the CGRP KO mice 6 h post‐inflammation, when acute nociceptive behavior was no longer observed. Furthermore, the bilateral tactile allodynia 6 h post‐inflammation was significantly decreased in the CGRP KO mice. In contrast, the acute nociceptive behavior immediately after the formalin injection was reduced only at 20–25 min post‐injection in the CGRP KO mice. These results suggest that endogenous CGRP contributes to peripheral inflammation‐induced synaptic plasticity in the amygdala, and this plasticity may underlie the exaggerated nociception–emotion linkage in pain chronification.     

EGF stimulates müller glial proliferation via a BMP‐dependent mechanism

Müller glia, the major type of glia in the retina, are mitotically quiescent under normal conditions, though they can be stimulated to proliferate in some pathological states. Among these stimuli, EGF is known to be a potent mitogen for Müller glia. However, the signaling pathways required for EGF‐mediated proliferation of Müller glia are not clearly understood. In this study, postnatal day 12 (P12) or adult trp53^?/? mouse retinas were explanted and cultured in the presence of EGF to stimulate Müller glial proliferation. Treatment with signaling inhibitors showed that activation of both MEK/ERK1/2 and PI3K/AKT pathways is required for EGF‐induced proliferation of Müller glia. Interestingly, BMP/Smad1/5/8 activation downstream of PI3K/AKT signaling was also necessary for robust Müller glial proliferation, though activation of BMP/Smad1/5/8 signaling alone failed to stimulate their proliferation. In dissociated Müller glial culture, treatment with EGF induced the upregulation of Bmp7, and this upregulation was blocked significantly by co‐treatment with the BMP inhibitor dorsomorphin, suggesting that BMP/Smad1/5/8 activation is mediated at least in part by an autocrine mechanism in Müller glia. A better understanding of how BMP/Smad1/5/8 signaling is involved in glial proliferation may have important implications for proliferative disorders, as well as for retinal regeneration in mammalian retinas.     

TrkB‐mediated activation of the phosphatidylinositol‐3‐kinase/Akt cascade reduces the damage inflicted by oxygen–glucose deprivation in area CA3 of the rat hippocampus

The selective vulnerability of hippocampal area CA1 to ischemia‐induced injury is a well‐known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen–glucose deprivation–reperfusion (OGD‐RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain‐derived neurotrophic factor (BDNF) or 7,8‐dihydroxyflavone (7,8‐DHF) exhibited reduced depression of the electrical activity triggered by OGD‐RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD‐RP. The protective effect of TrkB activation was limited to area CA3, as OGD‐RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3‐kinase/Akt (PI‐3‐K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD‐RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase‐3 activity in slices pretreated with BDNF and exposed to OGD‐RP. We propose that PI‐3‐K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.     

Smad3‐dependent signaling underlies the TGF‐β1‐mediated enhancement in astrocytic iNOS expression

We previously demonstrated that transforming growth factor‐β1 (TGF‐β1), while having no effect alone, enhances nitric oxide (NO) production in primary, purified mouse astrocytes induced by lipopolysaccharide (LPS) plus interferon‐γ (IFN‐γ), by recruiting a latent population of astrocytes to respond, thereby enhancing the total number of cells that express Nos2. In this investigation, we evaluated the molecular signaling pathway by which this occurs. We found that purified murine primary astrocytes express mRNA for TGFβRII as well as the TGFβRI subunit activin‐like kinase 5 (ALK5), but not ALK1. Immunofluorescence microscopy confirmed the expression of TGFβRII and ALK5 protein in astrocytes. Consistent with ALK5 signaling, Smad3 accumulated in the nucleus of astrocytes as early as 30 min after TGF‐β1 (3 ng/mL) treatment and persisted upto 32 hr after TGF‐β1 administration. Addition of ALK5 inhibitors prevented TGF‐β1‐mediated Smad3 nuclear accumulation and NO production when given prior to the Nos2 induction stimuli, but not after. Finally, astrocyte cultures derived from Smad3 null mutant mice did not exhibit a TGF‐β1‐mediated increase in iNOS expression. Overall, this data suggests that ALK5 signaling and Smad3 nuclear accumulation is required for optimal enhancement of LPS plus IFNγ‐induced NO production in astrocytes by TGF‐β1. © 2010 Wiley‐Liss, Inc.     

Tumor necrosis factor‐alpha mediates activation of NF‐κB and JNK signaling cascades in retinal ganglion cells and astrocytes in opposite ways

Tumor necrosis factor‐alpha (TNF) is an important mediator of the innate immune response in the retina. TNF can activate various signaling cascades, including NF‐κB, nuclear factor kappa B (NF‐κB) and c‐Jun N‐terminal kinase (JNK) pathways. The harmful role of these pathways, as well as of TNF, has previously been shown in several retinal neurodegenerative conditions including glaucoma and retinal ischemia. However, TNF and TNF‐regulated signaling cascades are capable not only of mediating neurotoxicity, but of being protective. We performed this study to delineate the beneficial and detrimental effects of TNF signaling in the retina. To this end, we used TNF‐treated primary retinal ganglion cell (RGC) and astrocyte cultures. Levels of expression of NF‐κB subunits in RGCs and astrocytes were evaluated by quantitative RT‐PCR (qRT‐PCR) and Western blot (WB) analysis. NF‐κB and JNK activity in TNF‐treated cells was determined in a time‐dependent manner using ELISA and WB. Gene expression in TNF‐treated astrocytes was measured by qRT‐PCR. We found that NF‐κB family members were present in RGCs and astrocytes at the mRNA and protein levels. RGCs failed to activate NF‐κB in the presence of TNF, a phenomenon that was associated with sustained JNK activation and RGC death. However, TNF initiated the activation of NF‐κB and mediated transient JNK activation in astrocytes. These events were associated with glial survival and increased expression of neurotoxic pro‐inflammatory factors. Our findings suggest that, in the presence of TNF, NF‐κB and JNK signaling cascades are activated in opposite ways in RGCs and astrocytes. These events can directly and indirectly facilitate RGC death.     

Zn2+‐induced ERK activation mediates PARP‐1‐dependent ischemic‐reoxygenation damage to oligodendrocytes

Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca²⁺]_i, loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia‐like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia‐induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O₂ (reperfusion‐like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high‐affinity Zn²⁺ chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high‐affinity Zn²⁺ probe, we showed that ischemia induced an intracellular Zn²⁺ rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia‐induced oligodendrocyte death. Moreover, UO126 blocked the ischemia‐induced increase in poly‐[ADP]‐ribosylation of proteins, and the poly[ADP]‐ribose polymerase 1 (PARP‐1) inhibitor DPQ significantly inhibited ischemia‐induced oligodendroglial cell death—demonstrating that PARP‐1 was required downstream in the Zn²⁺‐ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn²⁺, blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia‐reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult. © 2012 Wiley Periodicals, Inc.     

Neuroprotective effects of brain‐derived neurotrophic factor against amyloid beta 1‐40‐induced retinal and optic nerve damage

Brain‐derived neurotrophic factor (BDNF) could be considered a potential neuroprotective therapy in amyloid beta (Aβ)‐associated retinal and optic nerve degeneration. Hence, in this study we investigated the neuroprotective effect of BDNF against Aβ1‐40‐induced retinal and optic nerve injury. In this study, exposure to Aβ1‐40 was associated with retinal and optic nerve injury. TUNEL staining showed significant reduction in the apoptotic cell count in the BDNF‐treated group compared with Aβ1‐40 group. H&E‐stained retinal sections also showed a striking reduction in neuronal cells in the ganglion cell layer (GCL) of retinas fourteen days after Aβ1‐40 exposure. By contrast, number of retinal cells was preserved in the retinas of BDNF‐treated animals. After Aβ1‐40 exposure, visible axonal swelling was observed in optic nerve sections. However, the BDNF‐treated group showed fewer changes in optic nerve; axonal swelling was less frequent and less marked. In the present study, exposure to Aβ was associated with oxidative stress, whereas levels of retinal glutathione (GSH), superoxide dismutase (SOD) and catalase were significantly increased in BDNF‐treated than in Aβ1‐40‐treated rats. Both visual object recognition tests using an open‐field arena and a Morris water maze showed that BDNF improved rats’ ability to recognise visual cues (objects with different shapes) after Aβ1‐40 exposure, thus demonstrating that the visual performance of rats was relatively preserved following BDNF treatment. In conclusion, intravitreal treatment with BDNF prevents Aβ1‐40‐induced retinal cell apoptosis and axon loss in the optic nerve of rats by reducing retinal oxidative stress and restoring retinal BDNF levels.     

Impaired propulsive motility in the distal but not proximal colon of BK channel β1‐subunit knockout mice

Background Large‐conductance Ca²⁺‐activated K⁺ (BK) channels regulate smooth muscle tone. The BK channel β1‐subunit increases Ca²⁺ sensitivity of the α‐subunit in smooth muscle. We studied β1‐subunit knockout (KO) mice to determine if gastrointestinal (GI) motility was altered. Methods Colonic and intestinal longitudinal muscle reactivity to bethanechol and colonic migrating motor complexes (CMMCs) were measured in vitro. Gastric emptying and small intestinal transit were measured in vivo. Colonic motility was assessed in vivo by measuring fecal output and glass bead expulsion time. Myoelectric activity of distal colon smooth muscle was measured in vitro using intracellular microelectrodes. Key Results Bethanechol‐induced contractions were larger in the distal colon of β1‐subunit KO compared to wild type (WT) mice; there were no differences in bethanechol reactivity in the duodenum, ileum, or proximal colon of WT vsβ1‐subunit KO mice. There were more retrogradely propagated CMMCs in the distal colon of β1‐subunit KO compared to WT mice. Gastrointestinal transit was unaffected by β1‐subunit KO. Fecal output was decreased and glass bead expulsion times were increased in β1‐subunit KO mice. Membrane potential of distal colon smooth muscle cells from β1‐subunit KO mice was depolarized with higher action potential frequency compared to WT mice. Paxilline (BK channel blocker) depolarized smooth muscle cells and increased action potential frequency in WT distal colon. Conclusions & Inferences BK channels play a prominent role in smooth muscle function only in the distal colon of mice. Defects in smooth muscle BK channel function disrupt colonic motility causing constipation.     

Inhibition of cysteine cathepsin B and L activation in astrocytes contributes to neuroprotection against cerebral ischemia via blocking the tBid‐mitochondrial apoptotic signaling pathway

The roles of cathepsins in the ischemic astrocytic injury remain unclear. Here, we test the hypothesis that activation of cathepsin B and L contributes to the ischemic astrocyte injury via the tBid‐mitochondrial apoptotic signaling pathways. In the rat models of pMCAO, CA‐074Me or Clik148, a selective inhibitor of cathepsin B or cathepsin L, reduced the infarct volume, improved the neurological deficits and increased the MAP₂ and GFAP levels. In OGD‐induced astrocyte injury, CA‐074Me or Clik148 decreased the LDH leakage and increased the GFAP levels. In the ischemic cortex or OGD‐induced astrocytes injury, Clik148 or CA‐074Me reversed pMCAO or OGD‐induced increase in active cathepsin L or cathepsin B at 3 h or 6 h, increase in tBid, reduction in mitochondrial cytochrome‐c (Cyt‐c) and increase in cytoplastic Cyt‐c and active caspase‐3 at 12–24 h of the late stage of pMCAO or OGD. CA‐074Me or Clik148 also reduced cytosolic and mitochondrial tBid, increased mitochondrial Cyt‐c and decreased cytoplastic Cyt‐c and active caspase‐3 at 6 h of the early stage of Bid activation. CA‐074Me or Clik148 blocked the pMCAO‐induced release of cathepsin B or L from the lysosomes into the cytoplasm and activation of caspase‐3 in ischemic astrocytes at 12 h after ischemia. Concurrent inhibition of cathepsin B and cathepsin L provided better protection on the OGD‐induced astrocytic apoptosis than obtained with separate use of each inhibitor. These results suggest that inhibition of the cysteine cathepsin B and cathepsin L activation in ischemic astrocytes contributes to neuroprotection via blocking the tBid‐mitochondrial apoptotic signaling pathway. GLIA 2014;62:855–880     

Critical role of calpain in axonal damage-induced retinal ganglion cell death

Calpain, an intracellular cysteine protease, has been widely reported to be involved in neuronal cell death. The purpose of this study is to investigate the role of calpain activation in axonal damage-induced retinal ganglion cell (RGC) death. Twelve-week-old male calpstatin (an endogenous calpain inhibitor) knockout mice (CAST KO) and wild-type (WT) mice were used in this study. Axonal damage was induced by optic nerve crush (NC) or tubulin destruction induced by leaving a gelatin sponge soaked with vinblastine (VB), a microtubule disassembly chemical, around the optic nerve. Calpain activation was assessed by immunoblot analysis, which indirectly quantified the cleaved α-fodrin, a substrate of calpain. RGCs were retrogradely labeled by injecting a fluorescent tracer, Fluoro-Gold (FG), and the retinas were harvested and flat-mounted retinas prepared. The densities of FG-labeled RGCs harvested from the WT and CAST KO groups were assessed and compared. Additionally, a calpain inhibitor (SNJ-1945, 100 mg/kg/day) was administered orally, and the density of surviving RGCs was compared with that of the vehicle control group. The mean density of surviving RGCs in the CAST KO group was significantly lower than that observed in the WT group, both in NC and in VB. The mean density of surviving RGCs in the SNJ-1945-treated group was significantly higher than that of the control group. The calpain inhibitor SNJ-1945 has a neuroprotective effect against axonal damage-induced RGC death. This pathway may be an important therapeutic target for preventing this axonal damage-induced RGC death, including glaucoma and diabetic optic neuropathy and other CNS diseases that share a common etiology.     

The role of Homer1c in metabotropic glutamate receptor‐dependent long‐term potentiation

Group I metabotropic glutamate receptors (mGluR1/5) play a role in synaptic plasticity and they demonstrate direct interactions with the neuronal Homer1c protein. We have previously shown that Homer1c can restore the plasticity deficits in Homer1 knockout mice (H1‐KO). Here, we investigated the role of Homer1c in mGluR‐dependent synaptic plasticity in wild‐type mice, H1‐KO, and H1‐KO mice overexpressing Homer1c (KO+H1c). We used a form of plasticity induced by activation of mGluR1/5 that transforms short‐term potentiaion (STP) induced by a subthreshold theta burst stimulation into long‐term potentiation (LTP). We have shown that although acute hippocampal slices from wild‐type animals can induce LTP using this stimulation protocol, H1‐KO only show STP. Gene delivery of Homer1c into the hippocampus of H1‐KO mice rescued LTP to wild‐type levels. This form of synaptic plasticity was dependent on mGluR5 but not mGluR1 activation both in wild‐type mice and in KO+H1c. mGluR1/5‐dependent LTP was blocked with inhibitors of the MEK‐ERK and PI3K‐mTOR pathways in KO+H1c mice. Moreover, blocking Homer1c–mGluR5 interactions prevented the maintenance of LTP in acute hippocampal slices from KO+H1c. These data indicate that Homer1c–mGluR5 interactions are necessary for mGluR‐dependent LTP, and that mGluR1/5‐dependent LTP involves PI3K and ERK activation. © 2013 Wiley Periodicals, Inc.