Astrocytic Contribution to Motor Recovery After Spinal Cord Injury

Spinal cord injury results in loss of motor, sensory, and autonomic functions. Astrocytes, a cell type in the central nervous system, react to injury in a process called astrogliosis that impacts repair. At the site of injury, reactive astrocytes form an astrocytic scar that yields neuroprotective effects. Impaired formation of this scar causes increased tissue damage along with worsened motor recovery. Our lab identified leucine zipper-bearing kinase (LZK) as a key activator of astrogliosis that promotes wound healing after spinal cord injury in mice. This project examines the effect of astrocyte-specific LZK gene manipulation on hind-limb motor recovery following spinal cord injury. To determine the role of LZK on functional recovery post-injury, a complete crush was performed at thoracic level T8. Hindlimb function was measured using i) Basso Mouse Scale, an open field test that assesses gross motor function; and ii) regular horizontal ladder test that measures skilled stepping over 2 months after injury. Analysis demonstrated decreased function following LZK gene deletion. Decreased gross and fine motor function improvement was seen for the LZK-knock out when compared to the control genotype, suggesting that LZK is necessary for functional recovery. This is further suggested through increased gross motor function observed for the LZK-over expression when compared to the control genotype. Further research must be done to determine the role of LZK-over expression in fine motor recovery

Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS.

Reactive astrocytes influence post-injury recovery, repair, and pathogenesis of the mammalian CNS. Much of the regulation of astrocyte reactivity, however, remains to be understood. Using genetic loss and gain-of-function analyses in vivo, we show that the conserved MAP3K13 (also known as leucine zipper-bearing kinase [LZK]) promotes astrocyte reactivity and glial scar formation after CNS injury. Inducible LZK gene deletion in astrocytes of adult mice reduced astrogliosis and impaired glial scar formation, resulting in increased lesion size after spinal cord injury. Conversely, LZK overexpression in astrocytes enhanced astrogliosis and reduced lesion size. Remarkably, in the absence of injury, LZK overexpression alone induced widespread astrogliosis in the CNS and upregulated astrogliosis activators pSTAT3 and SOX9. The identification of LZK as a critical cell-intrinsic regulator of astrocyte reactivity expands our understanding of the multicellular response to CNS injury and disease, with broad translational implications for neural repair

The Anaphase-Promoting Complex or Cyclosome Supports Cell Survival in Response to Endoplasmic Reticulum Stress

The anaphase-promoting complex or cyclosome (APC/C) is a multi-subunit ubiquitin ligase that regulates exit from mitosis and G1 phase of the cell cycle. Although the regulation and function of APC/CCdh1 in the unperturbed cell cycle is well studied, little is known of its role in non-genotoxic stress responses. Here, we demonstrate the role of APC/CCdh1 (APC/C activated by Cdh1 protein) in cellular protection from endoplasmic reticulum (ER) stress. Activation of APC/CCdh1 under ER stress conditions is evidenced by Cdh1-dependent degradation of its substrates. Importantly, the activity of APC/CCdh1 maintains the ER stress checkpoint, as depletion of Cdh1 by RNAi impairs cell cycle arrest and accelerates cell death following ER stress. Our findings identify APC/CCdh1 as a regulator of cell cycle checkpoint and cell survival in response to proteotoxic insults

Characterization of the ER stress checkpoint in mammalian cells

Progression through the cell cycle adapts to both internal and environmental stimuli to ensure fidelity of cell division. Endoplasmic reticulum (ER) stress arising from an imbalance between cellular demand for protein folding and ER capacity has been described to cause G1 cell cycle arrest. The molecular components of this ER stress checkpoint have just begun to be uncovered. Although evidence emerge to suggest a prosurvival role for the ER stress-induced cell cycle arrest, the functional significance of this checkpoint in mammalian cells largely remains as an open question. Given the implication of ER stress in multiple human diseases like cancer and neurological disorders, elucidation of the link between ER stress and cell cycle may provide new insights to the pathogenesis and treatment of these diseases. In Chapter 1, I introduce the principles of cell cycle regulation and checkpoint responses, leading to a discussion on the discoveries that support an emerging ER stress checkpoint in eukaryotic cells. In Chapter 2, I investigate the mechanisms underlying cell cycle delay in G1 in response to ER stress in mammalian cells, showing that ER stress reduces the protein expression of Skp2 by downregulating Ufd1, a protein that stabilizes Skp2 through its ability to recruit the deubiquitinating enzyme USP13. This results in an accumulation of p27 that partly contributes to G1 arrest in ER-stressed cells. In Chapter 3, I identify another regulator of the ER stress checkpoint, APC/C-Cdh1, and begin to examine the upstream signals responsible for activating APC/C-Cdh1 under ER stress conditions. In Chapter 4, I provide a summary of the work and discuss the implications of my finding

The partial characterization of Cucurbita foetidissima (the buffalo gourd)

Digitized by Kansas Correctional Industrie

Ultrasensitive Gas Refractometer Using Capillary-Based Mach–Zehnder Interferometer

In this paper, we report a capillary-based Mach–Zehnder (M–Z) interferometer that could be used for precise detection of variations in refractive indices of gaseous samples. The sensing mechanism is quite straightforward. Cladding and core modes of a capillary are simultaneously excited by coupling coherent laser beams to the capillary cladding and core, respectively. An interferogram would be generated as the light transmitted from the core interferes with the light transmitted from the cladding. Variations in the refractive index of the air filling the core lead to variations in the phase difference between the core and cladding modes, thus shifting the interference fringes. Using a photodiode together with a narrow slit, we could interrogate the fringe shifts. The resolution of the sensor was found to be ~5.7 × 10−8 RIU (refractive index unit), which is comparable to the highest resolution obtained by other interferometric sensors reported in previous studies. Finally, we also analyze the temperature cross sensitivity of the sensor. The main goal of this paper is to demonstrate that the ultra-sensitive sensing of gas refractive index could be realized by simply using a single capillary fiber rather than some complex fiber-optic devices such as photonic crystal fibers or other fiber-optic devices fabricated via tricky fiber processing techniques. This capillary sensor, while featuring an ultrahigh resolution, has many other advantages such as simple structure, ease of fabrication, straightforward sensing principle, and low cost

Cdh1 depletion sensitizes cells to ER stress-induced cell death.

<p>(A) Empty vector-transfected or Cdh1-KD cells were treated with DMSO or 0.5, 1, or 2 µg/ml of TM for 9 h or 24 h. Total cell lysates were immunoblotted for cleaved PARP and indicated endogenous proteins. (B) Empty vector-transfected or Cdh1-KD cells were treated with DMSO, 0.5 µg/ml, or 1 µg/ml of TM for 9 h or 12 h. Total cell lysates were analyzed as in (A). (C) Empty vector-transfected or Cdh1-KD cells were treated with solvent (mock) or 1–3 mM DTT for 9 h or 12 h. Total cell lysates were immunoblotted as in (A). (D) <i>Left</i>, empty vector-transfected or Cdh1-KD cells were collected at 9, 12, and 24 h after treatment with DMSO or 0.5 µg/ml of TM for DNA content analysis by flow cytometry. Graph shows percentage of sub-G1 population at each time point. <i>Right</i>, empty vector-transfected or Cdh1-KD cells were collected at 9, 12, and 24 h after treatment with solvent (mock) or 1 mM DTT for DNA content analysis by flow cytometry. Graph shows percentage of sub-G1 population at each time point.</p

Mechanisms supporting APC/C^Cdh1 activity under ER stress conditions.

<p>(A) HeLa cells were treated with DMSO, 0.5 µg/ml of TM alone, or 0.5 µg/ml of TM plus 5 µM of MG-132 for 16 h. Immunoprecipitates of endogenous Cdc27 were immunoblotted for endogenous Cdh1. Immunoprecipitation using IgG served as negative control. ** indicates Cdh1-specific top band; *indicates non-specific bottom band. (B) HeLa cells were treated with DMSO or 1 µg/ml of TM for 16 h. CDK2 or CDK1 antibodies were used to immunoprecipitate endogenous CDK2 or CDK1 complexes, respectively. Immunoprecipitates were then used in <i>in vitro</i> kinase assays using histone H1 as substrate. <i>Left</i>, autoradiography of ³²P-histone H1 phosphorylated by CDK2 complexes. Coomassie stains input of histone H1 in the reactions. Intensity of the autoradioactive bands were quantified by Image J, normalized to histone H1 input, and presented as arbitrary units (A.U.). Immunoblot shows comparable levels of endogenous CDK2 and the indicated proteins before and after TM treatment. <i>Right</i>, autoradiography of ³²P-histone H1 phosphorylated by CDK1 complexes, analyzed as indicated for CDK2. (C) <i>Left</i>, total cell lysates from HeLa cells treated with DMSO or 1 µg/ml of TM for 16 h were immunoblotted for the indicated endogenous proteins. <i>Right</i>, quantification of endogenous Emi1 mRNA levels in HeLa cells treated with DMSO or 1 µg/ml of TM for 16 h, measured by SYBR-green qRT-PCR.</p

APC/C^Cdh1 is activated under ER stress conditions.

<p>(A) <i>Left</i>, HeLa cells were treated with DMSO or 2.5 µg/ml of tunicamycin (TM) for 8 h. Total cell lysates were immunoblotted for the indicated endogenous proteins. GRP78 served as a marker of ER stress. <i>Right</i>, HeLa cells were harvested at the indicated times after addition of DMSO or 2.5 µg/ml of TM. Total cell lysates were immunoblotted for the indicated endogenous proteins. HSP90 was used as a loading control. (B) HeLa cells were treated with DMSO or 1 µg/ml of tunicamycin for the indicated times. <i>Immunoblots</i>, total cell lysates were immunoblotted for the indicated endogenous proteins. <i>Graphs</i>, transcript levels as measured by SYBR-green qRT-PCR and protein levels as quantified by LiCOR-Odyssey software on immunoblots are compared for the indicated proteins. All measurements were normalized to the DMSO-0 h sample with relative value of 1. (C) HeLa cells were transfected with pSUPER (empty vector) or pSUPER-Cdh1-shRNA (Cdh1-KD). 24 h after transfection, cells were treated with DMSO, 1 µg/ml of TM alone, or 1 µg/ml of TM plus MG-132 (2 or 5 µM) for 16 h. Total cell extracts were immunoblotted for the indicated endogenous proteins.</p