Inducing Ubiquitylation and Protein Degradation as a Drug Development Strategy.

A new drug discovery strategy by inducing the degradation of oncoproteins through ubiquitin-proteasome system (UPS) has gained a lot of traction in the last decade (Verma et al. Mol Cell 77(3):446-460, 2020; Huang, Dixit. Cell Res 26:484, 2016). Multiple degrader platforms, such as IMiDs (Kronke et al. Science 343:301-305, 2014; Lu et al. Science 343:305-309; 2014), PROTAC (proteolysis targeting chimera) (Winter et al. Science 348:1376-1381, 2015), and molecular glues (Tan et al. Nature 446:640-645, 2007), have been approved or currently being developed in clinical trials. Compared to conventional drug inhibitors, degraders have a lot of advantages, such as catalytic mechanisms of action (MOA), no requirement of high-affinity ligands with targets, and potentially more sustained efficacy (Verma et al. Mol Cell 77(3):446-460, 2020; Huang, Dixit. Cell Res 26:484, 2016; Bondeson et al. Nat Chem Biol 11:611-617). Here, we describe protocols that measure intrinsic protein ubiquitination, degrader-induced target protein degradation, and cancer cell proliferation evaluation, as these protocols can help evaluate the potential of a drug target using a degrader platform.

Fluoxetine and its metabolite norfluoxetine induce microglial apoptosis.

Inflammatory insult to the central nervous system (CNS) can lead to development of depression, and subsequently depression is the most frequent psychiatric comorbidity following ischemic stroke, often limiting recovery and rehabilitation in patients. The initiators of inflammatory pathways in the CNS are microglia activated in response to acute ischemic stress, and anti-depressants have been shown to have anti-inflammatory effects in the CNS, promoting neuronal survival following ischemic insult. We have previously shown that the selective serotonin reuptake inhibitors (SSRIs) fluoxetine and citalopram promote neuronal survival after oxygen-glucose deprivation, an in vitro model of ischemia, by attenuating the release of glutamate and D-serine from activated microglia. Interestingly, we found that fluoxetine-treated microglial cultures contained fewer numbers of cells compared to other groups and hypothesized that fluoxetine and citalopram attenuated the release of glutamate and D-serine by promoting the apoptosis of microglia. The present study aimed to test and compare antidepressants from three distinct classes (tricyclics, monoamine oxidase inhibitors, and SSRIs) on microglial apoptosis. Primary microglia were treated with 1 µg/mL lipopolysaccharide and/or 10 µM antidepressants, and various apoptotic markers were assayed. Fluoxetine and its metabolite norfluoxetine decreased protein levels in cell lysates, decreased cell viability of microglia, and increased the expression of the apoptotic marker cleaved-caspase 3 in microglia. Live/dead nuclear staining also showed that fluoxetine- or norfluoxetine-treated cultures contained greater numbers of dying microglial cells compared to vehicle-treated cultures. Cultures treated with citalopram, phenelzine, or imipramine showed no evidence of inducing microglial apoptosis. Our results demonstrate that fluoxetine and norfluoxetine induce the apoptotic death of microglia, which may serve as a mechanism to attenuate the release of glutamate and D-serine from activated microglia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Neonatal rat microglia derived from different brain regions have distinct activation responses.

The regional heterogeneity of neuronal phenotypes is a well-known phenomenon. Whether or not glia derived from different brain regions are phenotypically and functionally distinct is less clear. Here, we show that microglia, the resident immune cells of the brain, display region-specific responses for activating agents including glutamate (GLU), lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP). Primary microglial cultures were prepared from brainstem (Brs), cortex (Ctx), hippocampus (Hip), striatum (Str) and thalamus (Thl) of 1-day-old rats and were shown to upregulate the release of nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) in a region- and activator-specific manner. With respect to ATP specifically, ATP-induced changes in microglial tumor necrosis factor-α (TNF-α) release, GLU uptake and purinergic receptor expression were also regionally different. When co-cultured with hypoxia (Hyp)-injured neurons, ATP-stimulated microglia from different regions induced different levels of neurotoxicity. These region-specific responses could be altered by pre-conditioning the microglia in a different neurochemical milieu, with taurine (TAU) being one of the key molecules involved. Together, our results demonstrate that microglia display a regional heterogeneity when activated, and this heterogeneity likely arises from differences in the environment surrounding the microglia. These findings present an additional mechanism that may help to explain the regional selectiveness of various brain pathologies.

Differential expression of PKC isoforms in developing zebrafish.

Protein kinase C isozymes are a biologically diverse group of enzymes known to be involved in a wide variety of cellular processes. They fall into three families (conventional, novel and atypical) depending upon their mode of activation. Several classes of zebrafish neurons have been shown to express PKCalpha during development, but the expression of other isoforms remains unknown. In this study we performed immunohistochemistry to determine if zebrafish express various isoforms of PKC. We used antibodies to test for the presence of enzymes that are thought to be preferentially expressed in the nervous system (PKCgamma, betaII, delta, epsilon, theta and zeta). Here, we show that PKCgamma, epsilon, theta and zeta are expressed in the zebrafish CNS. Anti-PKCgamma labels Rohon-Beard sensory neurons and Mauthner cells. PKCepsilon and zeta staining is widespread in the CNS, and PKCtheta and betaII are expressed in skeletal muscle, especially at intersegmental boundaries. Immunoblot experiments confirm the specificity of the antibodies in zebrafish and indicate that the fish isoforms of PKCgamma, betaII, epsilon and zeta are similar to the mammalian isoforms. Interestingly, PKCtheta appears to be similar to PKCthetaII, which, to date, has been found exclusively in mouse testis, but not in the mammalian CNS. Overall, our findings indicate that several different PKC isoforms are expressed in zebrafish, and that Rohon-Beard, Mauthner cells and muscle fibers preferentially express some isoforms over others.