Downregulation of hnRNPK mimics the “Abi-1 depletion phenotype” in neurons.

<p>(A) hnRNPK-RNAi knockdown in NIH3T3 cells with a construct targeting the 3′UTR-region of hnRNPK. (I) hnRNPK is endogenously expressed in NIH3T3, HeLA and Cos7 cells and is detectable in cultured hippocampal neurons (DIV21). (II) After transfection of NIH3T3 cells with an hnRNPK-RNAi construct for 3 days, the cells were fixed and stained with an antibody against hnRNPK. Only untransfected cells in close proximity to the RNAi-transfected cell in the upper right are immunopositive for hnRNPK, with a predominant distribution of the protein in the nucleus. (III) After transfection of NIH3T3 cells with an hnRNPK-RNAi construct for 3 days, protein expression of hnRNPK is markedly suppressed as confirmed by Western blotting compared to vector control transfected cells. (IV) Double transfections using two different hnRNPK-RNAi constructs, one targeting the 3′UTR of the hnRNPK sequence and one targeting the coding sequence of hnRNPK together with an hnRNPK-Myc-construct which is resistant against RNAi due to 4 nucleotide exchanges in the RNA leading to an unaltered amino acid sequence. The staining against the Myc-tag shows a decreased protein level solely when using the non-resistant construct together with the RNAi targeting the CDS (coding sequence). (B) Neuronal transfection of Abi-1-RNAi and hnRNPK-RNAi constructs. (I) In contrast to the control vector (pSuper), transfection of RNAi constructs resulted in an obvious change of neuronal morphology. The downregulation of hnRNPK as well as Abi-1 is leading to an extended and extremely branched dendritic tree. (II) The number of branching points within the dendritic compartment is significantly upregulated in both RNAi groups compared to the control transfection. (III) The analysis of the dendritic tree shows a significant shift of dendrites towards small, filopodia-like tertiary dendrites. Scale bars are as indicated.</p

Downregulation of hnRNPK reduces the number of mature synaptic contacts in hippocampal neurons.

<p>(AI,II) Decrease in mature synaptic contacts after Abi-1- or hnRNPK-RNAi knockdown. There is a significant decrease in the number of mature synapses with bassoon-positive presynaptic counterparts in the neurons transfected with Abi-1- or hnRNPK-RNAi constructs compared to vector control. At the same time, a more filopodia-like phenotype of the dendritic tree can be observed after RNAi knockdown of Abi-1 or hnRNPK. The RNAi resistant construct is able to rescue the observed reduction. Scale bars are as indicated.(BI,II) Analysis of the reduction of excitatory synapses by using ProSAP2/Shank3 antibodies that label postsynaptic densities (PSDs) reveals a comparable reduction of postsynaptic specializations that is also rescued by the RNAi resistant construct.</p

Expression pattern of hnRNPK in the developing CNS.

<p>(A) In situ hybridization of hnRNPK mRNA during rat brain development. At embryonic time points (1d), the mRNA of hnRNPK can easily be detected in all areas of the developing brain including the spinal cord. At later stages of maturation (horizontal sections, 3d-adult), the expression levels decrease and become more and more restricted to the cortex (Co), the hippocampal formation (Hc) and the granular layer of the cerebellum (Ce). (B) Immunohistochemical detection of hnRNPK in rat brain sagittal sections. At early time points of brain development (3d), a predominant nuclear labeling of hnRNPK can be detected in nearly all neurons. Again, cortex, hippocampus and cerebellum are most strongly labeled. At later time points, differences in spatial expression become even more prominent and intense staining is especially seen in granule cells of the cerebellum (Ce) and the dentate gyrus as well as in the CA1-4 regions of the hippocampus. In the cortex (Co), the staining intensity diminishes at later stages and only some scattered neurons in deeper cortical layers remain positive for hnRNPK. (C) Analysis of hnRNPK expression in different tissues and organs. hnRNPK is readily detectable in brain, liver and skeletal muscle (SM) while heart as well as lung, kidney, stomach and duodenum are almost devoid of detectable hnRNPK. (D) The analysis of hnRNPK expression in different brain areas (9d). A comparable expression profile of the protein is seen in the prefrontal cortex (PFC), the parietal cortex (Co), hippocampus (Hc), striatum (Str), thalamus (Tha), mesencephalon (Mes), and in the brain stem (Bst) while cerebellum (Ce) shows highest expression levels. Loading control: Actin. (E) Western blot analysis of time dependent hnRNPK expression in selected brain regions. hnRNPK detection in cerebellum, cortex and hippocampus at different stages of maturation shows that in all regions investigated, the strong signal at 8 to 28d becomes slightly weaker at 3 M. Loading control: Actin. Scale bars are as indicated.</p

hnRNPK interacts with Abi-1 via its KH2 domain.

<p>(A) Yeast two-hybrid screen. The full length Abi-1 cDNA was cloned as bait to screen a human fetal brain cDNA-library for putative interaction partners. 9 independent partial C-terminal hnRNPK clones were identified and retested for interaction by a yeast two-hybrid assay. Results are shown for the longest (aa248–464) and shortest (aa267–464) prey clone. hnRNPK is a 464 aa long protein that codes for several specific domains: N-terminal NLS, <i>nuclear localization signal</i>, KH1-KH3, <i>K homology domains 1–3 (light grey)</i>; KI, <i>K interaction domain (black)</i>; KNS, <i>K nuclear shuttling signal</i>. Abi-1 (476 aa) codes for the following domains: WAB, <i>WAVE binding domain</i>; SNARE, HHR, <i>homeobox homology region</i>; PP, <i>proline rich domain</i>; SH3 <i>src homology 3 domain</i>. (B) Schematic illustration of the Abi-1 and hnRNPK clones (and abbreviations) that have been used for further experiments. (C) The hnRNPK KH2 domain colocalizes with Abi-1.Several partial GFP- or Myc-tagged hnRNPK and Abi-1 clones were coexpressed in Cos7 cells to identify the interacting subdomains of the two proteins. In single transfection experiments, hnRNPK full length protein predominantly localizes to the nucleus, whereas Abi-1 shows a typical cytoplasmic staining pattern. When coexpressed, both proteins are localized in identical dotted structures (I). In contrast, the K1-recombinant protein alone is restricted to the nucleus and does not colocalize with Abi-1 after cotransfection (II). K2 fusion protein readily colocalizes with full-size Abi-1 in the cytoplasm (III). As shown for K1, K3 also shows no colocalization with Abi-1 (IV). The cotransfection of hnRNPK K2 with Abi-1 missing the SH3 domain (AbiΔSH3) results in no colocalization (V), the expression of Abi-1 SH3 domain alone (AbiSH3), however, gives rise to a perfect overlay in the perinuclear region (VI). (D) Coimmunoprecipitation experiments with overexpressed and endogenous Abi-1 and hnRNPK proteins. Plasmids encoding full-size hnRNPK-GFP and Abi-1-Myc were cotransfected in Cos7 cells and Abi-1-Myc was immobilized using anti-Myc microbeads loaded on a column. Protein-complexes then were eluted, separated by SDS-Page and hnRNPK-GFP (size 95 kDA) was detected by immunoblot using a specific anti-GFP antibody (I). As controls, beads loaded with lysate only (ctrl) and the input lysate were used. (II) Cos7 cells were transfected with partial hnRNPK-coding constructs K1-GFP (KH1 domain), K2-GFP (KH2 and KI domain), K3-GFP (KH3 domain) and K-full-GFP (full length) as GFP-fusion proteins. The correct expression of the hnRNPK constructs was controlled by using an anti-GFP antibody and a commercial anti-hnRNPK antibody that could detect the GFP fusion protein as well as the endogenous hnRNPK in the lysate (95 kDA and 65 kDA). Moreover, the commercial antibody detects the K2 construct. The correct expression and antibody specificity of the Abi-1-Myc construct was tested by cotransfection with truncated hnRNPK constructs and subsequent immunoblotting with an anti-Myc antibody. Afterwards, precipitation was performed with GFP-tagged microbeads after cotransfection of Abi-1-Myc and hnRNPK constructs. The precipitates were subjected to immunostaining with an anti-Myc antibody. The Abi-1-Myc protein could only be detected within hnRNPK-K2-GFP precipitate but not within K1-GFP and K3-GFP precipitate or within the GFP-only and/or negative controls. (III) <i>Vice versa</i> experiments were done by coimmunoprecipitations using lysates of Cos7 cells cotransfected with a combination of full length hnRNPK-Myc (K-Myc) and AbiΔSH3-GFP or AbiSH3-GFP, respectively. The immunoprecipitation was performed using antibodies directed against GFP and immunoblot-detection was performed using anti-hnRNPK antibodies showing that expression of the Abi-1 SH3 domain is a prerequisite for protein binding. (IV) The hnRNPK antibody was used to precipitate the protein complex from brain lysate as well as from the synaptosomal fraction. In the Western blot, an antibody against Abi-1 could readily detect its antigen in the precipitate. As positive control, brain lysate or synaptosomal material was used (Input lane: 4% of the total lysate used for immunoprecipitation), a negative control was performed with unspecific IgG (ctrl IgG). Scale bars are as indicated.</p

uncropped and unadjusted Westernblots

uncropped and unadjusted Westernblo

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1) - Table 1

<p>The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1) - Table 1 </p

Deletion of GGA GAE domain but not VHS domain disrupts BACE1-GGA interaction in CoIPs.

<p>(A) HEK293 cells were co-expressed with BACE1-HA and either myc-tagged GGA_wt, -ΔVHS, -ΔGAT, or-ΔGAE constructs. Equal expression was controlled by Western blot (left panels). BACE1-HA was immunoprecipitated with anti-HA beads (Miltenyi Biotec), and precipitation and co-precipitation of the GGAs was controlled by Western blot (right panels). Deletion of the VHS domain of GGA 1, 2, or 3 did not disturb interaction with BACE1 compared with wt GGAs (Lanes 1+2). Likewise, deletion of the GAT domain had no effect on binding to BACE1 (Lane 3), whereas deletion of the GAE domain dramatically reduced binding of GGAs and BACE1 (Lane 4). (B) HEK293 cells were co-expressed with BACE1-cterm-HA and either myc-tagged GGA_wt, -ΔVHS, -ΔGAT, or-ΔGAE constructs. Equal expression was controlled by Western blot (left panels). BACE1-cterm-HA was immunoprecipitated with anti-HA beads (Miltenyi Biotec), and precipitation and co-precipitation of the GGAs was controlled by Western blot (right panels). Comparable to full-length BACE1, deletion of the VHS domain of GGA1, 2, or 3 did not disturb interaction with BACE1-cterm compared with wt GGAs (Lanes 1+2). Likewise, deletion of the GAT domain had no effect on binding to BACE1 (Lane 3), whereas deletion of the GAE domain dramatically reduced binding of GGAs and BACE1-cterm (Lane 4). (C) We tested whether BACE1 GGA1-GAE domain interaction is due to a direct binding of both proteins by using <i>in vitro</i> CoIP. Myc-tagged GGA1-wt, -ΔVHS, and-ΔGAE and HA-tagged BACE1 were expressed separately in HEK293 (left panel) and purified by immunoprecipitation using magnetic beads (Miltenyi Biotec) labeled with the corresponding antibodies (middle panel). Purified proteins were mixed and immunoprecipitated with anti-HA beads. Both GGA1-wt and GGA1-ΔVHS were co-precipitated by BACE1, but GGA1-ΔGAE was not, indicating direct binding of BACE1 and the GGA1-GAE domain.</p

GGA-dependent BACE1 sorting and APP processing.

<p><b>(A)</b> APP and BACE1 are transported to the cell surface via secretory pathways. Both can be internalized into endosomal compartments where acidic conditions promote APP cleavage by BACE1 and Aβ generation. APP cleavage products and BACE1 are either recycled to the cell surface or sorted into late endosomal/lysosomal parthway for degradation. Likewise, APP, C99 and β- and γ-secretase can be sorted into autophagosomes for degradation as reviewed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref061" target="_blank">61</a>]. Increased secretase activity in these compartments were described to enhance Aβ production and the acidic conditions also promote Aβ aggregation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref062" target="_blank">62</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref064" target="_blank">64</a>]. Therefore, disturbed lysosomal degradation leads to intracellular increase of Aβ and Aβ oligomers and might enhance the release into the extracellular space [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref065" target="_blank">65</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref066" target="_blank">66</a>]. (B) GGAs directly connect the TGN and the endosomal/lysosomal system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref009" target="_blank">9</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref011" target="_blank">11</a>] and thereby can bypass cell surface transport of BACE1 and APP-SorLA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref037" target="_blank">37</a>]. Increase of GGA levels result in increased levels of intracellular sAPPβ and decreased levels of extracellular sAPPs and Aβ indicating enhanced endosomal/lysosomal transport of BACE1 and APP. (C) Whereas, SorLA GGA interaction is dependent on GGAs VHS-domain and SorLAs DXXLL motif, interaction of GGAs and BACE1 is primarily mediated by GGAs GAE-domain.</p

Human GGA protein family members.

<p>The GGA proteins were discovered almost simultaneously by five laboratories studying different aspects of membrane trafficking [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref049" target="_blank">49</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref052" target="_blank">52</a>]. The protein family consists of three members: GGA1, GGA2, and GGA3. GGAs are recruited from the cytosol onto the Golgi where they mediate the transport of cargo to endosomes/lysosomes. GGAs consist of four domains: the N-terminal VHS domain (Vps27, Hrs, and STAM); the GAT domain (GGA and Tom1), which contains two predicted coiled-coil domains; the hinge region, which contains one or more clathrin-binding sites; and the GAE domain (gamma-adaptin ear homology). The VHS domain is responsible for cargo recognition and binding via the AC-LL motifs in the cargo [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref006" target="_blank">6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref033" target="_blank">33</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref053" target="_blank">53</a>]. Membrane recruitment is provided by the GAT domain through ARF-GTP binding. Recently, the GAT C-terminal domains of GGA1 and GGA3 (but not GGA2) have also been shown to bind ubiquitin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref054" target="_blank">54</a>]. A distinct set of accessory proteins can bind to the GAE domain of GGAs, including rabaptin-5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref055" target="_blank">55</a>], epsin R (Enthoprotin or Clint) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref056" target="_blank">56</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref059" target="_blank">59</a>], γ-synergin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref060" target="_blank">60</a>], p56, and p200 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129047#pone.0129047.ref060" target="_blank">60</a>]. Shown are the GGA deletion mutants used in this study (lower left panel). The antibodies used, directed against GGA1 (H215/Santa Cruz), GGA2 (BD), and GGA3 (BD), bind to the hinge regions of GGAs as indicated (upper left panel).</p

Deletion of GGA GAT and GAE but not VHS domain increases secretion of APP β-cleavage products.

<p>N2A cells were co-transfected with APP-GFP and BACE1-HA and either GGA1, 2, or 3_wt-myc or domain deletion mutants. After 24 h, sAPP and Aβ levels were measured in the conditioned media by using sAPP and Aβ ELISA kits (MesoscaleDiscovery). As shown previously, secretion of sAPPα and β were reduced in cells overexpressing GGA1, 2, or 3 _wt or-ΔVHS compared with controls (left panels). Additionally, Aβ40 and Aβ42 levels were also reduced in cells co-expressing GGA_wt or-ΔVHS. Deletion of the GGA-GAT domain completely abolished the effect of sAPPs as well as Aβ secretion. Deletion of the GGA1 GAE domain but not GGA2 or 3 significantly increased sAPPβ levels compared with that of GGA_wt. Likewise, Aβ levels were increased for all three GGA GAE mutants, indicating altered APP processing due to altered BACE1-GGA interaction. Experiments were carried out in triplicate; shown are the results of n = 3 independent experiments. Statistical analysis was performed by using Kruskal-Wallis ANOVA on ranks and multiple comparison (Dunn’s method) (*p<0.05).</p