Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer’s disease

International audienceBackground: The 5XFAD early onset mouse model of Alzheimer's disease (AD) is gaining momentum. Behavioral, electrophysiological and anatomical studies have identified age-dependent alterations that can be reminiscent of human AD. However, transcriptional changes during disease progression have not yet been investigated. To this end, we carried out a transcriptomic analysis on RNAs from the neocortex and the hippocampus of 5XFAD female mice at the ages of one, four, six and nine months (M1, M4, M6, M9). Results: Our results show a clear shift in gene expression patterns between M1 and M4. At M1, 5XFAD animals exhibit region-specific variations in gene expression patterns whereas M4 to M9 mice share a larger proportion of differentially expressed genes (DEGs) that are common to both regions. Analysis of DEGs from M4 to M9 underlines the predominance of inflammatory and immune processes in this AD mouse model. The rise in inflammation, sustained by the overexpression of genes from the complement and integrin families, is accompanied by an increased expression of transcripts involved in the NADPH oxidase complex, phagocytic processes and IFN-γ related pathways. Conclusions: Overall, our data suggest that, from M4 to M9, sustained microglial activation becomes the predominant feature and point out that both detrimental and neuroprotective mechanisms appear to be at play in this model. Furthermore, our study identifies a number of genes already known to be altered in human AD, thus confirming the use of the 5XFAD strain as a valid model for understanding AD pathogenesis and for screening potential therapeutic molecules

Role of the voltage-gated K+ Channel Kv4.2 in rat sympathetic neuron Af currents

Potassium currents play an essential role in integrating neuronal signals by modulating the excitability of neurons and determining the action potential waveforms and firing patterns. Previously, Cooper and colleagues characterized voltage-gated K currents in neonatal neurons from the rat superior cervical ganglia (SCG). They demonstrated a positive correlation between the appearance of fast-inactivated currents (IAf) and the mRNA levels for the Kv4.2 voltage-gated potassium channel gene. Particularly, in cultured postnatal day 1 (P1) SCG neurons, both IAf and Kv4.2 mRNA decrease to very low levels after 14 days in culture. However, mRNA levels for other Kv genes that could also be responsible for Af currents on SCG neurons remained to be determined. My hypothesis was that Kv4.2 encodes K channel subunits underlying IAf in sympathetic neurons. To test this hypothesis, I built an adenoviral construct with a Kv4.2 insert to manipulate Kv4.2 expression in these neurons and compare the electrophysiological and pharmacological profiles of native IAf and Kv4.2-induced currents. (Abstract shortened by UMI.

Accumulation of Wildtype and ALS-Linked Mutated VAPB Impairs Activity of the Proteasome

Cellular homeostasis relies on a tight control of protein synthesis, folding and degradation, in which the endoplasmic reticulum (ER) quality control and the ubiquitin proteasome system (UPS) have an instrumental function. ER stress and aberrant accumulation of misfolded proteins represent a pathological signature of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder caused by the selective degeneration of motoneurons in the brain and spinal cord. Mutations in the ERresident protein VAPB have been associated with familial forms of the disease. ALS-linked mutations cause VAPB to form cytoplasmic aggregates. We previously demonstrated that viral-mediated expression of both wildtype and mutant human VAPB (hVAPB) leads to an ER stress response that contributes to the selective death of motoneurons. However, the mechanisms behind ER stress, defective UPS and hVAPB-associated motoneuron degeneration remain elusive. Here, we show that the overexpression of wildtype and mutated hVAPB, which is found to be less stable than the wildtype protein, leads to the abnormal accumulation of ubiquitin and ubiquitin-like protein conjugates in non-human primate cells. We observed that overexpression of both forms of hVAPB elicited an ER stress response. Treatment of wildtype and mutated hVAPB expressing cells with the ER stress inhibitor salubrinal diminished the burden of ubiquitinated proteins, suggesting that ER stress contributes to the impairment of proteasome function. We also found that both wildtype and mutated hVAPB can associate with the 20S proteasome, which was found to accumulate at the ER with wildtype hVAPB or in mutant hVAP

Mutated hVAPB is degraded faster than wildtype hVAPB through a proteasome-dependent mechanism.

<p>(A) Western blot analysis of COS-7 cells transfected with the indicated expression vectors and treated or not with the protein biosynthesis inhibitor cycloheximide (CHX, 100 µg/ml) for 3, 6, 8 and 10 h and/or with the proteasome blocking agent MG-132 (10 µM) for 10 h. Actin served as a loading control. (B) hVAPB immunoreactive bands were quantified by densitometry and values were normalized to actin and expressed relative to values obtained in untreated cells. (C) Densitometric quantification of hVAPB levels in transfected cells following 10 h of treatment with CHX and MG-132. (D-E) Immunolabeling of hVAPB in transfected COS-7 cells treated for 12 h with MG-132 (5 µM)(D). The number of cells showing a perinuclear accumulation of hVAPB was determined 36 h after transfection with the indicated vectors (E). Scale bar, 20 µm. Results shown in (B), (C) and (E) are the mean values ± S.D of three independent experiments.</p

hVAPB^P56S partially colocalizes with ubiquitinated conjugates but increases the general ubiquitin levels in the cells.

<p>(A) When co-transfected with hVAPB^WT or hVAPB^P56S, GFP-Ubi forms cytoplasmic aggregates that occasionally (white arrow) colocalize with hVAPB^WT or hVAPB^P56S. (B) The GFP-tagged mutated ubiquitin Ubi_AA-GFP does not form detectable aggregates. Scale bar, 20 µm. (C) Ubiquitin immunoblot profile of COS-7 cells transfected with empty, hVAPB^WT and hVAPB^P56S vectors following differential detergent extraction. (D) Western blot analysis of total HA-tagged ubiquitin levels in cells expressing either form of hVAPB or hVAPA. In (C) and (D), protein extracts were prepared 36 h after transfection and actin was used as a loading control.</p

hVAPB-mediated ER stress contributes to the accumulation of proteasome substrates.

<p>(A) The immunoreactivity of CHOP in cells expressing hVAPB^WT and hVAPB^P56S was monitored by Western blotting (36 h after transfection). Thapsigargin treatment (for 16 h) was used as a positive control for ER stress-dependent CHOP upregulation. (B) Levels of BiP and phosphorylation status of IRE1 were examined by Western blotting 36 h following the transfection of cells with empty, hVAPB^WT and hVAPB^P56S plasmids. (C) Protein extracts of cells transfected with the proteasome reporters Ub-R-YFP and Ub^G76V-YFP and treated (or not) for 16 h with the ER stress inducer thapsigargin (10 µM) were subjected to Western blotting using anti-GFP (referred to as YFP), and anti-CHOP antibodies. (D) Quantification of the YFP immunoreactive bands (C) normalized to actin signals (arbitrary densitometry units). (E) Salubinal treatment (20 µm) diminished the accumulation of the proteasome reporter (YFP) as indicated by Western blotting of COS-7 cells co-transfectd with hVAPB^WT or hVAPB^P56S. (F) Differential detergent extraction and Western blot analysis of ubiquitin in cells expressing hVAPB^WT and hVAPB^P56S and treated or not with salubrinal (20 µM).</p

Wildtype and mutated hVAPB associate with components of the secretory pathway in non-human primate cells.

<p>(A–D) Thirty-six hours following transfection of COS-7 cells, hVAPB^WT and hVAPB^P56S mainly colocalize with components of the secretory pathway as demonstrated by the immunostaining of hVAPB with the ER marker KDEL (A), the COPI vesicle marker β-COP-CFP (B) and ERGIC marker ERGIC-53 (C). hVAPB^P56S forms cytoplasmic aggregates that colocalize with ER, COPI and ERGIC markers. Both hVAPB^WT and hVAPB^P56S seldom colocalize (white arrow) with the COPII marker Sec23-YFP (D). Scale bar, 20 µm.</p

Wildtype and mutated hVAPB associate with the proteasome.

<p>(A) Both hVAPB^WT and hVAPB^P56S colocalize with the alpha 5 subunit of the proteasome, as indicated by the double immunostaining of cells transfected with empty, hVAPB^WT and hVAPB^P56S vectors. Images were acquired with the same exposure time and camera settings. White arrows indicates non-transfected cells. Scale bar, 20 µm. (B–C) hVAPB^WT and hVAPB^P56S were immunoprecipitated from COS-7 cells transfected with hVAPB^WT, hVAPB^P56S, myc-tagged Sar1, myc-tagged Arf1, myc-tagged hSOD1 and hVAPA. Endogenous alpha 5 (B) or alpha 1–7 (C) subunits of the proteasome that co-immunoprecipitated with hVAPBs was detected by Western blotting using specific antibodies. hVAPB, myc (Sar1, Arf1 and hSOD1) and hVAPA input levels are shown. Immunoprecipitation of alpha 1–7 by Rpt2 served as a positive control.</p

Accumulation of hVAPB^WT leads to the formation of cytoplasmic inclusions.

<p>(A) Sequential detergent extraction of cellular proteins at different times following transfection of COS-7 cells with indicated an empty vector, hVAPB^WT and hVAPB^P56S expression vectors. (B) Accumulation of wildtype hVAPB leads to the formation of insoluble inclusions that disrupt ER structure as documented by the co-immunostaining of hVAPB with KDEL 72 h after transfection. Scale bar, 20 µm.</p

Overexpression of wildtype and mutated hVAPB impairs proteasome activity.

<p>Levels of proteasome YFP reporters in cells co-transfected (or not) for 36 h with hVAPB^WT, hVAPB^P56S, hVAPA, hSOD1 and Ub-R-YFP (A), Ub-G76V-YFP (B) or CD3δ-YFP (C) were examined by Western blotting using anti-GFP antibodies. (D) Cells were co-transfected with vectors encoding hVAPB^WT, hVAPB^P56S or hVAPA and HA-tagged Fat10, an ubiquitin-independent signal for proteasomal degradation. Protein extracts were prepared 36 h post-transfection, resolved by SDS-PAGE, Western blotted and probed with HA, hVAPB, hVAPA and actin antibodies.</p