Gait and behavior in an IL1β-mediated model of rat knee arthritis and effects of an IL1 antagonist.

Interleukin-1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1β over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra-articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β-infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra-articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over-expression is associated limb hypersensitivity and gait compensation. Intra-articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

Quality control autophagy: a joint effort of ubiquitin, protein deacetylase and actin cytoskeleton.

Autophagy has been predominantly studied as a nonselective self-digestion process that recycles macromolecules and produces energy in response to starvation. However, autophagy independent of nutrient status has long been known to exist. Recent evidence suggests that this form of autophagy enforces intracellular quality control by selectively disposing of aberrant protein aggregates and damaged organelles--common denominators in various forms of neurodegenerative diseases. By definition, this form of autophagy, termed quality-control (QC) autophagy, must be different from nutrient-regulated autophagy in substrate selectivity, regulation and function. We have recently identified the ubiquitin-binding deacetylase, HDAC6, as a key component that establishes QC. HDAC6 is not required for autophagy activation per se; rather, it is recruited to ubiquitinated autophagic substrates where it stimulates autophagosome-lysosome fusion by promoting F-actin remodeling in a cortactin-dependent manner. Remarkably, HDAC6 and cortactin are dispensable for starvation-induced autophagy. These findings reveal that autophagosomes associated with QC are molecularly and biochemically distinct from those associated with starvation autophagy, thereby providing a new molecular framework to understand the emerging complexity of autophagy and therapeutic potential of this unique machinery.

VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD.

VCP (VCP/p97) is a ubiquitously expressed member of the AAA(+)-ATPase family of chaperone-like proteins that regulates numerous cellular processes including chromatin decondensation, homotypic membrane fusion and ubiquitin-dependent protein degradation by the proteasome. Mutations in VCP cause a multisystem degenerative disease consisting of inclusion body myopathy, Paget disease of bone, and frontotemporal dementia (IBMPFD). Here we show that VCP is essential for autophagosome maturation. We generated cells stably expressing dual-tagged LC3 (mCherry-EGFP-LC3) which permit monitoring of autophagosome maturation. We determined that VCP deficiency by RNAi-mediated knockdown or overexpression of dominant-negative VCP results in significant accumulation of immature autophagic vesicles, some of which are abnormally large, acidified and exhibit cathepsin B activity. Furthermore, expression of disease-associated VCP mutants (R155H and A232E) also causes this autophagy defect. VCP was found to be essential to autophagosome maturation under basal conditions and in cells challenged by proteasome inhibition, but not in cells challenged by starvation, suggesting that VCP might be selectively required for autophagic degradation of ubiquitinated substrates. Indeed, a high percentage of the accumulated autophagic vesicles contain ubiquitin-positive contents, a feature that is not observed in autophagic vesicles that accumulate following starvation or treatment with Bafilomycin A. Finally, we show accumulation of numerous, large LAMP-1 and LAMP-2-positive vacuoles and accumulation of LC3-II in myoblasts derived from patients with IBMPFD. We conclude that VCP is essential for maturation of ubiquitin-containing autophagosomes and that defect in this function may contribute to IBMPFD pathogenesis.

Effects of neuronal PIK3C3/Vps34 deletion on autophagy and beyond.

PIK3C3/Vps34 plays important roles in the endocytic and autophagic pathways, both of which are essential for maintaining neuronal integrity. However, it is unclear how inactivating PIK3C3 may affect neuronal endosomal versus autophagic processes in vivo. We generated a conditional null allele of the Pik3c3 gene in mouse, and specifically deleted it in postmitotic sensory neurons. Subsequent analyses reveal several interesting and surprising findings.

Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.

The Toronto Word Pool: Norms for imagery, concreteness, orthographic variables, and grammatical usage for 1,080 words

Imagery and concreteness norms and percentage noun usage were obtained on the 1,080 verbal items from the Toronto Word Pool. Imagery was defined as the rated ease with which a word aroused a mental image, and concreteness was defined in relation to level of abstraction. The degree to which a word was functionally a noun was estimated in a sentence generation task. The mean and standard deviation of the imagery and concreteness ratings for each item are reported together with letter and printed frequency counts for the words and indications of sex differences in the ratings. Additional data in the norms include a grammatical function code derived from dictionary definitions, a percent noun judgment, indexes of statistical approximation to English, and an orthographic neighbor ratio. Validity estimates for the imagery and concreteness ratings are derived from comparisons with scale values drawn from the Paivio, Yuille, and Madigan (1968) noun pool and the Toglia and Battig (1978) norms. © 1982 Psychonomic Society, Inc.