Serum FAM19A5 in neuromyelitis optica spectrum disorders: Can it be a new biomarker representing clinical status?

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) targets astrocytes and elevates the levels of astrocyte-injury markers during attacks. FAM19A5, involved in reactive gliosis, is secreted by reactive astrocytes following central nervous system (CNS) damage. OBJECTIVE: To investigate the significance of serum FAM19A5 in patients with NMOSD. METHODS: We collected clinical data and sera of 199 patients from 11 hospitals over 21 months. FAM19A5 levels were compared among three groups: NMOSD with positive anti-aquaporin-4 antibody (NMOSD-AQP4), other CNS demyelinating disease, and healthy controls. RESULTS: The median serum FAM19A5 level was higher in the NMOSD-AQP4 (4.90 ng/mL (3.95, 5.79)) than in the other CNS demyelinating (2.35 ng/mL (1.83, 4.07), p < 0.001) or healthy control (1.02 ng/mL (0.92, 1.14), p < 0.001) groups. There were significant differences in the median serum FAM19A5 levels between the attack and remission periods (5.89 ng/mL (5.18, 6.98); 4.40 ng/mL (2.72, 5.13), p < 0.001) in the NMOSD-AQP4 group. Sampling during an attack (p < 0.001) and number of past attacks (p = 0.010) were independently associated with increased serum FAM19A5. CONCLUSION: Serum FAM19A5 was higher in patients with NMOSD-AQP4 and correlated with clinical characteristics. Thus, serum FAM19A5 may be a novel clinical biomarker for NMOSD-AQP4.

Disruption of polyubiquitin gene Ubc leads to attenuated resistance against arsenite-induced toxicity in mouse embryonic fibroblasts.

The polyubiquitin gene Ubc is upregulated under oxidative stress induced by arsenite [As(III)]. However, the detailed mechanism of Ubc upregulation and the exact role of ubiquitin (Ub) to protect cells against As(III)-induced toxicity remain unknown. Here, we found that Ubc-/- mouse embryonic fibroblasts (MEFs) exhibited reduced viability under As(III) exposure, although the Nrf2-Keap1 pathway was activated as a cytoprotective response. Intriguingly, due to the reduced polyubiquitination and delayed onset of degradation of Nrf2 in Ubc-/- MEFs, the basal expression levels of Nrf2 target genes were elevated. As(III)-induced accumulation of Ub conjugates occurred in an Nrf2-independent manner, probably due to cellular stress conditions, including reduced proteasomal activity. Increased cellular Ub levels were essential to polyubiquitinate misfolded proteins generated under As(III) exposure and to degrade them by the proteasome. However, when cellular Ub levels decreased, these misfolded proteins were not efficiently polyubiquitinated, but rather accumulated as large protein aggregates inside the cells, causing cytotoxicity. Furthermore, increased activity of the autophagic pathway to clear these aggregates was not observed in Ubc-/- MEFs. Therefore, reduced viability of Ubc-/- MEFs under As(III) exposure may not be due to dysregulation of the Nrf2-Keap1 pathway, but mostly to reduced efficacy to polyubiquitinate and degrade misfolded protein aggregates.

Restoration of cellular ubiquitin reverses impairments in neuronal development caused by disruption of the polyubiquitin gene Ubb.

Disruption of the polyubiquitin gene Ubb leads to hypothalamic neurodegeneration and metabolic disorders, including obesity and sleep abnormalities, in mice. However, it has yet to be determined whether or not these neural phenotypes in Ubb(-/-) mice are directly caused by cell autonomous defects in maintaining proper levels of ubiquitin (Ub). To directly demonstrate that reduced levels of Ub are sufficient to cause neuronal abnormalities, we investigated the characteristics of cultured neurons isolated from Ubb(-/-) mouse embryonic brains. We found that neuronal morphology, neurite outgrowth, and synaptic development were significantly impaired in Ubb(-/-) neurons. Furthermore, we observed the growth of astrocytes in Ubb(-/-) cell cultures despite the fact that cells were cultured under conditions promoting neuronal growth. When the reduced levels of free Ub, but not Ub conjugates, in Ubb(-/-) cells were restored to those of wild-type cells by providing exogenous Ub via lentivirus-mediated delivery, the increased apoptosis observed in Ubb(-/-) cells was almost completely abolished. Ectopic expression of Ub also improved neuronal and glial phenotypes observed in Ubb(-/-) cells. Therefore, our study suggests that Ub homeostasis, or the maintenance of cellular free Ub above certain threshold levels, is essential for proper neuronal development and survival.

Locus coeruleus neurons are resistant to dysfunction and degeneration by maintaining free ubiquitin levels although total ubiquitin levels decrease upon disruption of polyubiquitin gene Ubb.

Previously, we demonstrated that disruption of polyubiquitin gene Ubb leads to hypothalamic neurodegeneration and metabolic abnormalities associated with hypothalamic dysfunction. However, we cannot exclude the possibility that defects in other brain regions where Ubb is highly expressed may also contribute to the phenotypes exhibited by Ubb(-/-) mice. Upon searching for such brain regions, we identified a region in the brainstem called the locus coeruleus where both polyubiquitin genes Ubb and Ubc were highly expressed. In contrast to other brain regions, Ubc was significantly upregulated in the locus coeruleus of Ubb(-/-) mice presumably to compensate for loss of Ubb, and this upregulation was sufficient to maintain levels of free Ub, but not total Ub, in the locus coeruleus. However, in the hypothalamus of Ubb(-/-) mice, both free and total Ub levels significantly decreased. This discrepancy resulted in completely different phenotypic outcomes between the two different brain regions. While we have reported dysfunction and degeneration of hypothalamic neurons in adult Ubb(-/-) mice, there were no signs of functional impairment or degeneration in the locus coeruleus neurons, suggesting that the maintenance of free Ub above threshold levels could be an important mechanism for neuronal protection. Accordingly, we propose that, upon stress induced by disruption of Ubb, neuronal vulnerability may be determined based on the ability of neurons or neighboring cells to maintain free Ub levels for the protection of neuronal function and survival.

Quantification of oxidative stress in live mouse embryonic fibroblasts by monitoring the responses of polyubiquitin genes.

Stress-regulated polyubiquitin genes in mammals are expected to be upregulated under oxidative stress conditions. In order to assess gene regulation via the conventional method, the isolation of RNA molecules or the transfection of reporter constructs into cells is frequently required. If the stress response within cells can be monitored in a reversible manner with minimal manipulation, the study of the stress response pathways will become much easier. Herein, we have developed a simple fluorescence plate reader-based assay to monitor the stress responses of polyubiquitin genes in mouse embryonic fibroblasts, in which one allele of the ubiquitin-coding region of the polyubiquitin gene Ubb or Ubc was replaced by the eGFP-puro cassette, thereby placing GFP expression under the control of the endogenous polyubiquitin gene promoter. Using this simple assay, we established that both mammalian polyubiquitin genes are upregulated upon oxidative stress with slightly higher responses from the Ubb promoter. The principal advantage of this assay is that it allows for the monitoring of stress responses of polyubiquitin genes without disrupting cellular growth; this assay can therefore be applied repeatedly to the same cells. Furthermore, by calculating the increase in fluorescence deriving from newly synthesized GFP upon stress, which can be regarded as a bona fide polyubiquitin gene stress response, we were able to determine and directly compare the concentrations of various oxidative stressors that induce the similar cellular stress levels. Therefore, this simple assay may also be employed in the screening of potentially toxic reagents that induce the stress response pathways.

Disruption of polyubiquitin gene Ubb causes dysregulation of neural stem cell differentiation with premature gliogenesis.

Disruption of polyubiquitin gene Ubb leads to early-onset reactive gliosis and adult-onset hypothalamic neurodegeneration in mice. However, it remains unknown why reduced levels of ubiquitin (Ub) due to loss of Ubb lead to these neural phenotypes. To determine whether or not the defects in neurons or their progenitors per se, but not in their cellular microenvironment, are the cause of the neural phenotypes observed in Ubb(-/-) mice, we investigated the properties of cultured cells isolated from Ubb(-/-) mouse embryonic brains. Although cells were cultured under conditions promoting neuronal growth, Ubb(-/-) cells underwent apoptosis during culture in vitro, with increased numbers of glial cells and decreased numbers of neurons. Intriguingly, at the beginning of the Ubb(-/-) cell culture, the number of neural stem cells (NSCs) significantly decreased due to their reduced proliferation and their premature differentiation into glial cells. Furthermore, upregulation of Notch target genes due to increased steady-state levels of Notch intracellular domain (NICD) led to the dramatic reduction of proneuronal gene expression in Ubb(-/-) cells, resulting in inhibition of neurogenesis and promotion of gliogenesis. Therefore, our study suggests an unprecedented role for cellular Ub pools in determining the fate and self-renewal of NSCs.