Journal: Human Molecular Genetics

Article Title: IKKβ and mutant huntingtin interactions regulate the expression of IL-34: implications for microglial-mediated neurodegeneration in HD

doi: 10.1093/hmg/ddx315

Figure Lengend Snippet: Induction of IL-34 by mHTTx1 is mimicked by neuronal chemical stressors. (A) NPCs of each line were induced to differentiate and harvested at the indicated days post differentiation (DPD). Isolated RNAs were quantified for the levels of IL-34 mRNA using standard RT-qPCR with Taqman probes and normalized to GAPDH mRNA levels. (B) WB analysis of lysates from 8-day old neuronal cultures for levels of IL-34 protein. Glycosylated and unmodified forms of IL-34 with apparent molecular weights of ∼55 kDa and ∼27 kDa, respectively, can be seen to be selectively induced by Q-expanded mHTTx1(Q103) but not by control HTTx1(Q25). (C) DNA damage activates IL-34 transcription and exacerbates the effects of mHTTx1. 8-day old cultures were treated with vehicle (V) or 0.5 μM of etoposide (ETO) for 48 hrs. Extracted RNA was tested for IL-34 mRNA levels as in (A). (D) Control and IKKβ KD cultures were treated with NMDA or staurosporine (ST) for 8 hrs. Extracted RNA was tested for IL-34 as in (A). All experiments were repeated three times. Data are presented as means ± SD. ***P < 0.001. C=Control NPCs differentiated for the indicated days.

Article Snippet: Human IL-34 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_152456","term_id":"289666747","term_text":"NM_152456"}} NM_152456 ) was obtained from Origene (Rockville, MD).

Techniques: Isolation, Quantitative RT-PCR

Journal: Human Molecular Genetics

Article Title: IKKβ and mutant huntingtin interactions regulate the expression of IL-34: implications for microglial-mediated neurodegeneration in HD

doi: 10.1093/hmg/ddx315

Figure Lengend Snippet: IKKβ regulates mHTTx1-induced IL-34. (A) NPCs for each line were differentiated for the indicated days (DPD). RNA was extracted and IL-34 mRNA was quantified as in Figure 1A. All experiments were confirmed at least three times. Data are presented as means ± SD. ***P < 0.001. (B) Confirmation using WB analysis that IKKβ blocks induction of IL-34 by mHTTx1 at the protein level. C=Control NPCs differentiated for the indicated days.

Article Snippet: Human IL-34 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_152456","term_id":"289666747","term_text":"NM_152456"}} NM_152456 ) was obtained from Origene (Rockville, MD).

Techniques:

Journal: Human Molecular Genetics

Article Title: IKKβ and mutant huntingtin interactions regulate the expression of IL-34: implications for microglial-mediated neurodegeneration in HD

doi: 10.1093/hmg/ddx315

Figure Lengend Snippet: IKKβ inhibitors reduce mHTTx1 oligomerization and IL-34 production. (A) Inhibition of mHTTx1 protein aggregation by the small molecule IKKβ inhibitors luteolin and TPCA (top) and of IL-34 expression (middle) were determined by WB analysis. (B) Concentration-dependent inhibition of mHTTx1 aggregation by sodium salicylate (top) and of IL-34 expression (middle). MW8 was used to detect mHTTx1 aggregates and a rabbit anti-IL-34 was used to detect IL-34. (C) Luteolin was further examined for its effect on IL-34 mRNA expression by qRT-PCR. (C = control, Lut = luteolin, V= vehicle, TPCA = 2-[(aminocarbonyl) amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide). All experiments were repeated three times. Data presented are means ± SD. ***P < 0.001. (D) Sodium salicylate was also examined for its effect on IL-34 mRNA expression by qRT-PCR. Data presented are means ± SD. ***P < 0.001. The apparent IC50 for luteolin is ∼2 μg/ml and ∼100 μg/ml for sodium salicylate (Fig. 4C and ​andDD).

Article Snippet: Human IL-34 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_152456","term_id":"289666747","term_text":"NM_152456"}} NM_152456 ) was obtained from Origene (Rockville, MD).

Techniques: Inhibition, Expressing, Concentration Assay, Quantitative RT-PCR

Journal: Human Molecular Genetics

Article Title: IKKβ and mutant huntingtin interactions regulate the expression of IL-34: implications for microglial-mediated neurodegeneration in HD

doi: 10.1093/hmg/ddx315

Figure Lengend Snippet: IL-34 exacerbates mHTTx1-induced degeneration of MSNs. Corticostriatal brain slice explants were biolistically co-transfected with YFP and/or mHTTx1 expression constructs as described in the Materials and Methods to induce degeneration of striatal medium spiny neurons (MSNs). (A) Representative fluorescent micrographs of MSNs transfected with a sub-threshold amount of mHTTx1 showing no degeneration by 3 days in culture (compare upper left and right panels) unless further co-transfected with an expression construct for IL-34 (lower right panel). Co-transfection with IL-34 only had no obvious effects on MSNs viability (lower left panel). Scale bar, 200 μm. (B) Quantification of numbers of healthy MSNs per brain slice explants as identified by the co-transfected YFP marker in each condition as described in (A). Averaged means ± SD are shown for 4 independent experimental runs, with the YFP positive control condition set to 100%. *P <0.05 by ANOVA followed by Dunnett's post hoc comparison test. (C) Treatment of brain slice explants with the CSF1R inhibitor PLX3397 shows concentration-dependent inhibition of microglial numbers/activation as visualized using anti-Iba1 immunostaining. (D) In contrast, MSNs in corticostriatal brain slices transfected with a supra-threshold amount of mHTTx1 (twice the amount of mHTTx1 DNA loaded onto the biolistic gold particles as for Parts A and B above) undergo overt and significant neurodegeneration by 4 days after transfection (compare first two bars). Treatment with PLX3397 in the same concentration range that showed microglial inhibition (c.f. panel C) provided significant neuroprotection to mHTTx1-transfected MSNs. Averaged means ± SD are shown for five independent experimental runs, with the mHTTx1 DMSO-treated condition set to 100%; third bar shows positive control condition in which mHTTx1-transfected brain slices are treated with a cocktail of 50 µM KW-6002 and 30 µM SP600125. *P <0.05 by ANOVA followed by Dunnett's post hoc comparison test.

Article Snippet: Human IL-34 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_152456","term_id":"289666747","term_text":"NM_152456"}} NM_152456 ) was obtained from Origene (Rockville, MD).

Techniques: Slice Preparation, Transfection, Expressing, Construct, Cotransfection, Marker, Positive Control, Concentration Assay, Inhibition, Activation Assay, Immunostaining

Journal: Human Molecular Genetics

Article Title: IKKβ and mutant huntingtin interactions regulate the expression of IL-34: implications for microglial-mediated neurodegeneration in HD

doi: 10.1093/hmg/ddx315

Figure Lengend Snippet: Procaspase-3 induction by mHTTx1 aggregation is inhibited by IKKβ knockdown. (A) WB analysis of lysates from neurons expressing mHTTx1 (left lanes) and with IKKβ KD (right lanes) for the accumulation of procaspase-3. (B) IL-34 does not inhibit mHTTx1 aggregation or procaspase-3 induction. NPCs expressing mHTTx1 were co-cultured with 0, 5%, or 10% of another NPC line expressing IL-34 resulting in up to 10-fold increases in expression of secreted IL-34 protein in the co-cultures. Supernatants of 8-day-old cultures were quantified for IL-34 secretion by ELISA. All experiments were reproduced at least three times. Data presented are means ± SD. ***P <0.001. (C) WB analysis of neuronal lysates of co-cultures from (B) showing that neither mHTTx1 aggregation (top panel) nor procaspase-3 induction (middle panel) was affected by increased levels of soluble IL-34 in the co-cultures. N. S.= Non-specific.

Article Snippet: Human IL-34 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_152456","term_id":"289666747","term_text":"NM_152456"}} NM_152456 ) was obtained from Origene (Rockville, MD).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Human Molecular Genetics

Article Title: IKKβ and mutant huntingtin interactions regulate the expression of IL-34: implications for microglial-mediated neurodegeneration in HD

doi: 10.1093/hmg/ddx315

Figure Lengend Snippet: A model for mHTTx1-induced IL-34 signaling driving neurodegeneration. Accumulation of mHTTx1 creates a stressed neuronal environment activating IKKβ, which in turn promotes the aggregation of mHTTx1 and subsequent induction of procaspase-3 (ProCas-3) and IL-34. In turn, increased IL-34 secretion may initially signal to microglia to express neuroprotective factors and maintain survival. However, chronic elevation of IL-34 by neurons accumulating mHTT may lead instead to pathological activation of microglia, producing inflammatory cytokines and necrotic factors to drive neuronal degeneration and death via non-cell autonomous interactions.

Article Snippet: Human IL-34 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_152456","term_id":"289666747","term_text":"NM_152456"}} NM_152456 ) was obtained from Origene (Rockville, MD).

Techniques: Activation Assay