1 Isoform-specific subcellular localization and function of protein kinase A identified by
2 mosaic imaging of mouse brain
3 Ronit Ilouz1*, Varda Lev-Ram1, Eric A. Bushong2, Travis Stiles3, Dinorah Friedmann-
4 Morvinski4,5, Christopher Douglas3, Geoffrey Goldberg3, Mark H. Ellisman2,6, and Susan S.
5 Taylor1,7*
6 7 8 9 10 11 12 13
14 1Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.
15 2Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of 16 California, San Diego, La Jolla, CA 92093, USA. 17 18 3Department of Ophthalmology, Shiley Eye Center, University of California, San Diego, La Jolla, CA 92093, USA 19 20 4Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA 21
22 5Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv 23 University, Tel Aviv 69978, Israel
24 6Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, CA 92093, USA.
25 7Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA
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28 * Correspondence: [email protected], [email protected]
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1 30 ABSTRACT
31 Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different
32 regulatory (R) subunits. Deficiency in either RIβ or RIIβ subunits results in distinct neuronal
33 phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using
34 isoform-specific antibodies we generated high-resolution large-scale immunohistochemical
35 mosaic images of mouse brain that provide global views of several brain regions, including the
36 hippocampus and cerebellum. The isoforms concentrate in discrete brain regions and we then
37 zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-
38 localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron
39 microscopy we confirm mitochondrial and nuclear localization of RIβ in cultured neurons. To
40 show the functional significance of nuclear localization, we demonstrate that down-regulation of
41 RIβ, but not RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform
42 specificity is defined by precise localization.
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2 50 INTRODUCTION
51 Precise spatiotemporal regulation of signaling molecules is central to the intricacies of
52 signal transduction. cAMP-dependent protein kinase (PKA), ubiquitously expressed in every
53 mammalian cell, regulates numerous signaling pathways and is critical for many neuronal
54 functions. This includes learning and memory (Kandel, 2012) and multiple forms of synaptic
55 plasticity (Abel et al., 1997; Yasuda et al., 2003). Pharmacological or genetic inhibition of PKA
56 severely affects the induction of hippocampal long-term potentiation and inhibits synaptic
57 plasticity and long lasting memory (Abel et al., 1997). A reduction in PKA signaling contributes
58 to the etiology of several neurodegenerative diseases, including Alzheimer’s disease and
59 Parkinson’s disease (Dagda and Das Banerjee, 2015; Howells et al., 2000). Phosphorylation
60 mediated by cAMP signaling is critically involved in cell growth, differentiation, apoptosis,
61 synaptic release of neurotransmitters and gene expression (Skalhegg and Tasken, 2000), and a
62 large part of the functional diversity of this kinase results from isoform diversity. Eukaryotic
63 cells express multiple forms of PKA regulatory (R) and catalytic (C) subunits. PKA holoenzyme
64 consists of an R-subunit dimer bound to two C- subunits (R2C2). The biochemical and functional
65 features of PKA holoenzymes are largely determined by the structure and the biochemical
66 properties of the regulatory subunits (Ilouz et al., 2012; Taylor et al., 2012; Zhang et al., 2012).
67 There are two classes of R-subunits, RI and RII, which are classified into α and β
68 subtypes (Doskeland et al., 1993; McKnight et al., 1988). Each isoform is encoded by a unique
69 gene and preferentially expressed in different cells and tissues. RIα and RIIα are ubiquitously
70 expressed in every cell, whereas RIβ and RIIβ expression is more tissue restricted (Cadd and
71 McKnight, 1989). RIβ is expressed in brain and spinal cord (Cadd and McKnight, 1989). RIIβ is
72 predominantly expressed in brain, endocrine, fat, liver and reproductive tissues (Cadd and
3 73 McKnight, 1989; Jahnsen et al., 1986). The four R-subunits are functionally non-redundant.
74 Depletion of either RIβ or RIIβ gene in mice resulted in specific neuronal defects. RIIβ knockout
75 mice display defects in motor behavior and loss of PKA mediated neuronal gene expression
76 (Brandon et al., 1998). Hippocampal slices from RIβ null mice show a severe deficit in long-term
77 depression and depotentiation at the Schaffer collateral-CA1 synapse. Despite a compensatory
78 increase in the RIα protein levels, hippocampal function was not rescued (Brandon et al., 1995).
79 The composition and specific structural and biochemical properties of PKA holoenzymes
80 (R2C2) account, in part, for differential cellular responses to discrete extracellular signals that
81 activate adenylate cyclase (Taylor et al., 2012). Space-restricted kinase activation provides an
82 extra layer of specificity in PKA signaling. PKA is typically targeted at specific intracellular
83 microdomains through interactions with A-Kinase Anchoring Proteins (AKAPs). Many AKAPs
84 have been identified as well as their specific requirements for selective binding to regulatory
85 subunits (Sarma et al., 2010; Wong and Scott, 2004). This spatio-temporal regulation determines
86 the access of proteins to interacting binding partners. AKAPs provide a control mechanism to
87 direct, integrate and locally attenuate the cAMP-initiated cascade. The hallmark signature motif
88 of the AKAPs is an amphipathic helix that binds tightly to the dimerization and docking (D/D)
89 domain of the R-subunits. Recently, a point mutation in the D/D domain of the RIβ gene has
90 been associated with a new neurodegenerative disease presenting with dementia and
91 Parkinsonism, characterized by specific and abundant accumulation of RIβ in neuronal
92 inclusions (Wong et al., 2014).
93 Rigorous cellular characterization of protein localization is a necessary step if we aim to
94 understand PKA function in a physiological context. To date, relatively few efforts have
95 attempted to systematically define the subcellular localization of endogenous proteins using
4 96 imaging-based techniques. Currently, RIβ, the isoform that has a unique role in synaptic
97 plasticity (Brandon et al., 1995) and has been associated with a neurodegenerative disease, is the
98 least studied PKA isoform. RIβ spatial localization has not been systematically studied due to
99 antibody cross reactivity with RIα. Most of the available RIIβ localization data is focused,
100 furthermore, on specific regions of interest; thus the global context of the protein localization is
101 lost.
102 Subcellular cAMP signaling domains are defined by the distinct environments within
103 cellular organelles. The dogma of cAMP-PKA signaling in the nucleus states that, upon cAMP-
104 induced activation of the cytosolic PKA holoenzyme, the C-subunit dissociates from the R-
105 subunit in an isoform-specific manner and translocates into the nucleus via diffusion
106 (Harootunian et al., 1993). Contradictory, reports have increasingly proposed the existence of
107 resident pools of nuclear PKA holoenzyme (Jarnaess et al., 2009; Sample et al., 2012; Zippin et
108 al., 2004). While the necessity of a proper nuclear PKA activity for neuronal function is well-
109 documented the existence of PKA R-subunits or their physiological role within the nucleus has
110 not been well-studied.
111 In this study, we generated high-resolution large-scale mosaic images of several mouse
112 brain slices using RIβ and RIIβ-specific antibodies. Since RIβ is the least studied isoform at the
113 protein level, we focused our analyses on brain regions where we expected RIβ to be
114 predominant based on its mRNA expression profiles and its predicted functional importance
115 from RIβ(-/-) mice. The use of large-scale immunohistochemical brain maps allows us to gain an
116 overview of the RIβ and RIIβ protein distribution over large areas and then zoom in to obtain
117 higher resolution views in order to investigate subcellular features. We found that each
118 regulatory isoform is predominant in distinct brain regions and were able to identify unique and
5 119 consistent patterns of distribution within the hippocampus and the cerebellum. RIβ is
120 concentrated in dendrites, and co-localizes with MAP2, whereas RIIβ is concentrated in axons.
121 We confirmed the RIβ subcellular distribution that emerged from the mosaic images using the
122 mini-Singlet Oxygen Generator (miniSOG) labeling, a probe that allowed us to do correlated
123 light and electron microscopy. We found RIβ at the mitochondria, as we predicted earlier, as well
124 as the nucleus, establishing a new paradigm for PKA signaling in the nucleus. To demonstrate a
125 functional distinction between the two isoforms in the nucleus we selectively down regulated the
126 two isoforms and used CREB phosphorylation as a reporter for a nuclear PKA substrate. Down-
127 regulation of RIβ, but not RIIβ, decreased pCREB in hippocampal cultures. These
128 comprehensive brain images are accessible via Cell Centered Database (CCDB) to browse or
129 download.
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6 142 RESULTS
143 Overview of the regional distribution of RIβ and RIIβ across brain regions
144 The overall patterns of RIβ and RIIβ protein distribution across brain regions within a full
145 coronal slice are shown in Figure 1. Images were acquired using confocal microscopy. Image
146 stacks were then knit together to create high-resolution 2D large-scale brain images. The
147 resulting image mosaics provide detailed views of cellular and subcellular distribution of RIβ
148 and RIIβ without losing the context of the tissue. To map the protein distribution of RIβ and
149 RIIβ, we stained mouse brain sections with specific RIβ and RIIβ antibodies. Although RIIβ
150 antibody is commonly used and its specificity has been previously validated (Weisenhaus et al.,
151 2010), RIβ immunohistochemical imaging has not been possible previously due to the high cross
152 reactivity of existing antibodies with RIα. Thus, it was essential to confirm the absolute
153 specificity of the RIβ antibodies. To achieve high specificity we removed any RIα cross-reacting
154 antibodies with immobilized RIα protein as described in Material and Methods and then
155 extensively validated the specificity and selectivity of the RIβ and RIIβ antibodies (Figure 1-
156 figure supplement 1) Western blots confirmed the specificity of the antibodies for RIα vs. RIβ
157 purified proteins (Figure 1-figure supplement 1, upper left panel) while dot blots of the four
158 purified R-subunit isoforms demonstrated the specificity and selectivity of our RIβ and RIIβ
159 antibodies to the proteins in their native conformation (Figure 1-figure supplement 1, upper right
160 panel). To confirm specificity in cells we overexpressed RIβ-MKO2 or RIα-MKO2 in mouse
161 10T1/2 cells; RIβ antibody detects overexpressed RIβ-MKO2 but not the overexpressed RIα-
162 MKO2. Similar experiments were also done for the RIIβ antibody to validate its specificity and
163 selectivity (Figure 1-figure supplement 1). The antibody did not cross react with RIIα, its closest
164 homolog. Staining the overexpressed cells with secondary antibodies alone resulted in no signal
7 165 at similar levels or higher laser power, demonstrating no background from secondary antibodies
166 in our method (Figure 1-figure supplement 1, A4&C4). For an additional negative control, RIβ
167 antibody was incubated with purified full-length human RIβ protein. The pre-absorbed antibody
168 blocked the antibody’s specific binding and no signal was detected when tested on the RIβ-
169 MKO2 overexpressed cells or a brain slice (Figure 1-figure supplement 1 A5&C5, Figure 1-
170 figure supplement 2).
171 These mosaic maps allow us to identify the predominant isoform within different brain
172 regions across the sections, as defined by the brain atlas (Paxinos et al., 2001) We find that the
173 overall patterns of RIβ and RIIβ distribution are discrete. As shown in Figure 1A, RIβ is
174 expressed predominantly in the hippocampus, specifically in the CA1-3 pyramidal cell layers as
175 well as in the dentate gyrus. RIβ is also highly expressed at the medial habenular nuclei but not
176 at the lateral habenular nuclei of the forebrain. Strong signals are observed in the allocortex
177 including the piriform cortex and the retrosplenial granular cortex and much less in the
178 neocortex. RIβ is observed also in the dorsal endopiriform nucleus. In the hypothalamus, strong
179 cell body labeling is seen in the subincertal nucleus. Comparing RIβ protein levels to its
180 previously reported mRNA levels (Cadd and McKnight, 1989), we observe that RIβ mRNA
181 levels are higher in the pyramidal cell layer compared to the dentate gyrus but the protein is
182 expressed at similar levels in both regions.
183 In our images, RIIβ is predominantly expressed in the striatum (Figure 1B). RIIβ was
184 previously shown to be the major PKA isoform in the striatum, including the caudoputamen,
185 nucleus accumbens, and islands of calleja by in situ hybridization analysis (Cadd and McKnight,
186 1989) as well as by comparing the RIIβ protein expression from various dissected brain regions
187 (Brandon et al., 1998). RIIβ is also seen strongly at the choroid plexus and blood vessels. RIIβ
8 188 strongly labels the mossy fibers of the hippocampus but not the pyramidal cell layer of the
189 hippocampus. Increased labeling within specific nuclei of the thalamus and hypothalamus was
190 observed. Based on comparison with the Franklin and Paxinos mouse brain atlas, we posit that
191 substantial levels of RIIβ are also evident at the reticular thalamic nucleus, rhomboid and
192 reuniens thalamic nucleus. In the hypothalamus, ventromedial nuclear cells are strongly labeled
193 as well as the arculate nucleus in the mediobasal hypothalamus.
194 Differential expression of RIβ and RIIβ at the hippocampus
195 To investigate the differential expression pattern of RIβ and RIIβ in detail within any
196 specific region of interest we used the brain images to zoom in from the gross sectional levels to
197 the sub-neuronal levels of a specific region of interest. The images provide detailed views of
198 cellular and subcellular structures without losing the context of the brain region. The problem of
199 relating higher magnified views with the lower scale surveys is eliminated in this method. Figure
200 2A shows a detailed view of the hippocampus. RIβ is specifically localized to the pyramidal cell
201 layers of CA1-3 region. RIβ is localized to the cell bodies of these cells as well as their dendrites
202 including both basal dendrites and the apical dendrites (Figure 2B, upper panel). Interestingly,
203 RIβ, but not RIIβ, is localized to the hippocampal interneurons, which are the inhibitory neurons
204 (Figure 2B, lower panel). RIIβ is excluded from the pyramidal cell layer (Figure 2C). The protein
205 expression patterns seen in these maps correlate well with the mRNA detected by in situ
206 hybridization (Cadd and McKnight, 1989).
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9 210 RIIβ is concentrated in axons whereas RIβ is concentrated in dendrites and somata in
211 various subfields of the hippocampus.
212 We next utilized the large-scale mosaic images to explore detailed views of various
213 subfields within the hippocampus. The advantage of the high-resolution large-scale mosaic
214 images is that these comprehensive maps allow analyzing protein localization at gross structural
215 level and at the same time provide subcellular details, thereby allowing tracing an axonal
216 pathway and at the same time visualizing details such as axon terminals (Figure 3-figure
217 supplement 1). We can trace an axonal pathway, where RIIβ is enriched, that emerges from the
218 hilus of the dentate gyrus to the mossy fibers (Figure 3A, Figure 3-figure supplement 1). RIIβ is
219 abundant in the axons that emerge from the granule cells of the dentate gyrus and pass through
220 the hilus (Figure 3B). RIIβ is also concentrated in the mossy fiber axon terminals in the CA3-
221 stratum lucidum (Figure 3C). This is consistent with in situ hybridization data that shows that the
222 granule cells of the dentate gyrus express RIIβ mRNA abundantly (Cadd and McKnight, 1989).
223 RIβ localization is distinct at these regions as it is concentrated in the CA3 pyramidal cell layer
224 including the dendrites. RIIβ is abundant in the axons of the stratum lacunosum-moleculare layer
225 that contains perforant path fibers (Figure 3D). The advantage of the detailed hippocampus map
226 is that we can zoom in into different axons within the subfields such as the stratum lacunosum-
227 layer or stratum radiatum and identify synaptic boutons, which helps to assign the staining
228 specifically to axons (Figure 3-figure supplement 1). RIIβ is also enriched in the alveus, which is
229 the deepest layer that contains the axons from pyramidal neurons, passing on towards the
230 fimbria/fornix, one of the major outputs of the hippocampus (Figure 3E). The parallel path of
231 axonal fibers, immunostained by RIIβ, in the alveus is evident (Figure 3-figure supplement 1).
10 232 RIIβ axonal staining does not co-localizes with the RIβ dendritic staining. RIβ is excluded from
233 these axons.
234 This data is consistent with the RIIβ enrichment seen at the mossy fibers (Weisenhaus et
235 al., 2010), but is in contrast to the data provided by Zhong, H. et al (Zhong et al., 2009) where
236 RIIβ localization was analyzed at high resolution in dendritic shafts and spines using both
237 cultured neurons and an in vivo approach using transfected PKA subunits.
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239 RIβ and RIIβ distinct expression at the cerebellum
240 We generated additional maps to examine the overall protein distribution of RIβ and RIIβ
241 isoforms in the cerebellum and provide a full view of one folium (Figure 4). We focus on the
242 cerebellum because RIβ, the least studied PKA isoform, is the most predominant PKA isoform in
243 this region and no immunohistochemical studies were previously published. Western blot
244 analysis of the cerebellum fraction showed that RIβ was expressed in this region (Gronholm et
245 al., 2003) whereas RIIβ expression was not detected (Weisenhaus et al., 2010), probably due to
246 its low expression compared to the other regions detected. The in situ hybridization data showed
247 much less hybridization of RIIβ compared to RIβ at the cerebellum which is consistent with the
248 protein expression (Cadd and McKnight, 1989). A detailed comparison of the two isoforms
249 shows distinct localization patterns. RIβ is highly enriched in the Purkinje cells including the
250 somata and the dendritic trees (Figure 4B-C). RIβ is also localized to the granular cell layer and
251 excluded from the white matter (Figure 4D). RIIβ is preferentially expressed at the somata of the
252 Purkinje cells and is detected at lower levels at their dendrites, and is mainly at the primary
253 branches. The RIIβ expression is consistent with previous reports as well as AKAP150
254 localization, which is the primary AKAP responsible for targeting PKA to dendritic spines and
11 255 post-synaptic density (Glantz et al., 1992). RIβ is abundant at the cerebellar glomerulus compare
256 to RIIβ (Figure 4D).
257 Consistently, the full mosaic maps of the cerebellum and the hippocampus demonstrate
258 that dendrites are preferentially expressing the RIβ isoform at higher levels. RIβ is expressed at
259 the dendrites of the pyramidal cell layer in the hippocampus as well as at the dendritic trees of
260 the Purkinje cells at the cerebellum. RIIβ expression was excluded from the pyramidal cell layer
261 and was very weak at the dendrites of the Purkinje cells, suggesting a unique role for RIβ at cell
262 dendrites.
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264 RIβ co-localizes with MAP2 at the cerebellum
265 The subcellular localization of PKA regulatory subunits is typically controlled by A
266 kinase anchoring proteins (AKAPs) (Wong and Scott, 2004). Microtubule associated protein 2
267 (MAP2), a dendritic marker, is also known as a dendritic AKAP that plays a substantial role in
268 type II PKA localization in dendritic shafts (Theurkauf and Vallee, 1982). Since RIβ is
269 concentrated in dendrites and abundantly expressed in the cerebellum compared to RIIβ, we
270 performed co-localization experiments with MAP2 in this region. Figure 5A shows that in
271 cerebellum slices RIβ co-localizes with MAP2. The RIIβ isoform, that has been shown to
272 interact with MAP2 in the striatum and hippocampus, is less concentrated in the cerebellum and
273 co-localizes with MAP2 only at the dendritic branches of the Purkinje cell (Figure 5B). In
274 hippocampal/cortical primary cultured cells, we show that RIβ co-localizes with MAP2, and
275 consistent with previous reports RIIβ also partially co-localizes with MAP2 in these cells (Figure
276 5C-D). Although more experiments need to be done to verify direct interactions between RIβ and
277 MAP2, our study suggests that MAP2 may contribute to RIβ anchoring to dendrites.
12 278 RIβ is a nuclear PKA isoform in neuronal cells
279 While the translocation of the catalytic subunit into the nucleus and the phosphorylation
280 of nuclear factors have been well characterized, the function of nuclear regulatory subunits
281 remains largely unexplored. In our brain maps we typically find RIβ in the nucleus whereas RIIβ
282 is excluded from the nucleus. Examples of the RIβ nuclear localization are shown in Figure 6.,
283 and include the Purkinje cells of the cerebellum (Figure 6A), interneurons in the hippocampus
284 (Figure 6B), and neuronal cells in the hypothalamus (Figure 6C). We confirmed that the
285 secondary antibodies do not provide non-specific background on the brain slice (Figure 6-figure
286 supplement 1). In primary cultured cells of hippocampal/cortical neurons RIβ is also found at the
287 cell nucleus, whereas RIIβ is excluded from the nucleus (Figure 6D). Stimulation of these cells
288 with forskolin to activate adenylate cyclases did not result in RIIβ translocation to the nucleus
289 (data not shown). The molecular processes that direct translocation of the R-subunits to the
290 nucleus are as yet undefined.
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292 RIβ subcellular localization identified by electron microscopy
293 To further assess and to define more precisely the nuclear localization of RIβ observed by the
294 fluorescent images we used electron microscopy and the recently introduced miniSOG labeling
295 (Shu et al., 2011). MiniSOG is a genetically modified flavoprotein, which produces singlet
296 oxygen upon excitation. DAB will then be oxidized to form an insoluble osmium-philic polymer
297 which gives contrast to the ultrastructure near the tagged fusion protein. RIβ cDNA was fused to
298 miniSOG and electrophorated into primary hippocampal cells. As shown in Figure7D we find
299 that RIβ is localized to the mitochondria as we previously reported by cell fractionation
300 experiments (Ilouz et al., 2012). The resolution allows us to more specifically localize RIβ to the
13 301 cristae and the inner membrane of the mitochondria. A control untranfected photo-oxidized
302 mitochondria is shown in Figure 7C. 126 transfected darkened mitochondria and 71 non-
303 transfected light mitochondria were counted. A representative image for a non-transfected
304 (Figure 7C) and a transfected (Figure 7D) mitochondrion is shown. Moreover, we can detect RIβ
305 localization in the cell nucleus confirming the fluorescent images of brain sections (Figure 7B).
306 A control untransfected photo-oxidized cell nucleus is shown in Figure 7A. Consistent with this,
307 RIβ protein levels are much lower in the human brain cytosolic fraction as compared with the
308 particulate fraction. It is only the RIβ regulatory subunit among all four regulatory subunits that
309 show high protein levels in the particulate fractions of postmortem brains (Chang et al., 2003).
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311 RIβ, but not RIIβ, contributes PKA-dependent CREB phosphorylation
312 One of the nuclear targets of PKA is the transcription factor cyclic AMP response
313 element-binding protein (CREB). While the necessity for PKA activation and a subsequent
314 phosphorylation of CREB on Ser133 is well documented for many neuronal signaling pathways
315 including synaptic plasticity (Kandel, 2012), the specific physiological function of PKA
316 regulatory subunits in these signaling complexes are only beginning to be understood. Since we
317 find RIβ in the nucleus we sought to investigate whether down-regulation of RIβ would result in
318 impairment of CREB phosphorylation, a nuclear PKA substrate, in cultured cells. To accomplish
319 this, we designed lentiviral shRNA vectors containing GFP to knockdown RIβ or RIIβ subunits.
320 Thus, cells expressing shRNA plasmids can be identified by the presence of GFP. We first
321 confirmed the knockdown efficiency by co-transfecting 293T cells with cDNA encoding either
322 RIβ or RIIβ genes, together with a scrambled shRNA (shRNA IRR) as a negative control or
323 shRNAs targeting either RIβ or RIIβ. Western blot analysis confirmed knockdown efficiency of
14 324 RIβ at the protein level (Figure 8-figure supplement 1A). Western blot analysis indicated that an
325 efficient RIIβ knockdown had been achieved using shRNA RIIβ clone #2 (Figure 8-figure
326 supplement 1B). This clone was used in the subsequent experiments to knockdown RIIβ. To
327 further validate the knockdown efficiency of these shRNA lentiviruses in primary cultured
328 neurons we infected the cells with shRNAs targeting either RIβ or RIIβ and used scrambled
329 shRNA (shRNA SCR) as a negative control. Cells were immunostained with either RIβ or RIIβ
330 specific antibodies. Infected cells expressing the indicated shRNAs were identified by the
331 presence of GFP. Cells expressing shRNA against RIβ or RIIβ display a significant reduction in
332 the cell fluorescence intensity produced by their specific antibodies as compared to cells infected
333 with a negative control. The results indicate a significant inhibition of RIβ or RIIβ expression in
334 cells infected with shRNA against a corresponding gene. Representative cells are shown in
335 Figure 8-figure supplement 1C. The shRNAs against RIβ or RIIβ demonstrate 70% or 80%
336 knockdown efficiency, respectively, in neuronal cells (P-value<0.0001) (Figure 8-figure
337 supplement 1D).
338 Following knockdown validation, we sought to investigate whether down-regulation of
339 RIβ would result in impairment of CREB phosphorylation. We infected dissociated rat
340 hippocampal cultures with GFP-shRNA lentiviruses against RIβ, RIIβ or an irrelevant control.
341 After recovery from infection, cells were stimulated with forskolin, an adenylyl cyclase
342 activator, to increase intracellular levels of cAMP and activate PKA. The physiological
343 consequence of RIβ and RIIβ down-regulation for nuclear cAMP signaling was evaluated by
344 analysis of CREB phosphorylation at Ser 133. Due to the robust survival capacity of non-
345 neuronal cells, trace gila present in initial preparations were an unavoidable component of these
346 cultures. Non-infected cells remain as well. To specifically assay CREB phosphorylation in
15 347 infected cells, the signal was quantified only in cells positively stained for βIII-tubulin (neuronal
348 marker, red) that expressed GFP (marker for lentivirus infection). CREB phosphorylation was
349 quantified in the specified cells as a measure of pCREB signal intensity per the area of the
350 nucleus as delineated by DAPI. We find that in forskolin-stimulated neuronal cells, a significant
351 reduction in the intensity of phospho-Ser133 CREB was detected in neurons infected with
352 shRNA against RIβ compared to the control (p<0.0001) (Figure 8A, Figure 8B). In contrast, no
353 significant decline in the intensity of phospho-Ser133 CREB was detected in cells infected with
354 shRNA against RIIβ (Figure 8A, Figure 8B). The decline in phospho-CREB in neurons infected
355 with shRNA against RIβ was also significant compared to neurons infected with shRNA against
356 RIIβ (p=0.0003).
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16 367 DISCUSSION
368 Signaling events mediated by PKA are critical for many neuronal functions. While the
369 necessity of proper PKA catalytic activity and the phosphorylation of its downstream substrates
370 are well established in a variety of neuronal functions, the rational and significance for having
371 multiple PKA regulatory isoforms has not been appreciated. In this study, we used highly
372 specific antibodies against RIβ and RIIβ to create high-resolution large-scale mosaic images of
373 the brain, and these images reveled distinct patterns of localization. Although RIβ is expressed at
374 low levels in most cells, it is enriched in the brain, and our images show that RIβ, but not RIIβ, is
375 highly expressed in the hippocampus, consistent with tissue fractionations and with many of its
376 predicted roles in neuronal function. In spite of its importance for neuronal function RIβ has
377 never been comprehensively imaged before due to the lack of specific antibodies. Furthermore,
378 there has never been a systematic comparison of these two isoforms that are abundantly
379 expressed in the brain. These mouse brain mosaic images thus provide detailed global
380 comparative views of whole brain sections and also help to explain the functional non-
381 redundancy of RIβ and RIIβ. In addition, the maps allow one to interrogate subcellular features
382 without losing the context of the brain regions. By zooming in to different brain regions such as
383 the hippocampus and cerebellum we consistently find that RIβ is enriched in dendrites and co-
384 localizes with MAP2 whereas RIIβ is more concentrated in axons. In our mosaic maps, RIβ is
385 also localized to the nucleus within different brain regions. To verify the subcellular localization
386 at higher resolution we used miniSOG labeling, a technique that allows us to correlate light
387 microscopy with electron microscopy. Consistent with our earlier report we found RIβ at the
388 mitochondria (Ilouz et al., 2012), and with the miniSOG labeling we could further localize RIβ to
389 the inner membrane and cristae of the mitochondria. In addition, RIβ was prominently seen in
17 390 the nucleus and around the nuclear envelope. To demonstrate a functional distinction between
391 the two isoforms at the nucleus, we used primary neuronal cells and specific lenti-viruses to
392 selectively down regulate RIβ or RIIβ and then tracked CREB phosphorylation as a reporter for a
393 PKA nuclear substrate. Down-regulation of the RIβ, but not RIIβ, decreased CREB
394 phosphorylation, which is consistent with the observed nuclear localization of RIβ.
395 Our mosaic maps provide an overview of the distribution of endogenous PKA regulatory
396 subunits in a whole brain slice and at the same time allow us to identify subcellular features. The
397 distinct subcellular localization of PKA RIβ and RIIβ subunits within discrete brain regions
398 emphasizes the importance of mapping spatial localization of proteins in the context of the tissue
399 whereas cultured cells sometimes do not provide an accurate representation of spatial
400 localization. We find that each regulatory subunit is predominant within different brain regions
401 and has a distinct and consistent patterns of cellular and subcellular localization across multiple
402 regions. This isoform-specific localization provides an additional layer for achieving specificity
403 in PKA signaling, and identifies localized pools of PKA signaling complexes in the brain. The
404 specific pattern of distribution of RIβ in the hippocampus is consistent with its known unique
405 roles in synaptic plasticity (Brandon et al., 1995). RIβ but not RIIβ is predominant at the
406 pyramidal neurons in the CA1-CA3 regions of the hippocampus, as well as in the dentate gyrus
407 and hilus neuron (Figure 1). This localization is consistent with hippocampal slices from RIβ
408 null mice, which showed a severe deficit in LTD and depotentiation at the Schaffer collateral-
409 CA1 synapse. This defect was also evident at the lateral perforant path-dentate granule cell
410 synapse in RIβ knockout mice (Brandon et al., 1995). In contrast, and also consistent with
411 previous reports, our maps show that RIIβ is not expressed at the pyramidal neurons but is
412 concentrated in the striatum. In correlation with the RIIβ expression pattern, targeted disruption
18 413 of the RIIβ gene in mice lead to a dramatic down-regulation of total PKA activity in the striatum
414 and a partial compensatory increase in RI subunits (Brandon et al., 1998).
415 Nuclear signaling events mediated by PKA are critical for numerous neuronal functions
416 including activation of gene expression required for long term memory (Kandel, 2012). A
417 surprising and intriguing finding in this study is that RIβ is the PKA regulatory subunit that is
418 localized to the nucleus of many cells in different brain regions, and miniSOG labeling of RIβ
419 confirmed the nuclear localization at a nanoscale resolution by electron microscopy. Induction of
420 cAMP and activation of PKA leads to phosphorylation of specific transcription factors and
421 induction of gene expression and long-term changes in neurons (Greengard, 2001). Nuclear
422 localization of RI and RII has been reported in hepatocytes by immunogold labeling (Kuettel et
423 al., 1985). There is evidence suggesting that nuclear RIIβ binds CREB in T cells, implicating
424 nuclear RIIβ as a potential negative regulator of T cell activation (Elliott et al., 2003). Whether
425 PKA is resident in the nucleus or translocated into the nucleus in response to cytoplasmic signals
426 is still an open question. Clearly one now also needs to carefully explore all of the PKA isoforms
427 as well as specific cell types.
428 PKA holoenzymes are recruited into well-defined macromolecular signaling complexes
429 through a specific interaction between the dimerization and docking (D/D) domain, a conserved
430 hydrophobic domain within PKA R-subunits, and A Kinase Anchoring Proteins (AKAPs).
431 These scaffold proteins determine the spatio-temporal activity of PKA. Over 50 members and
432 splice variants of the AKAP family have been identified (Scott et al., 2013). While most AKAPs
433 were initially identified by their ability to bind RII subunits, several AKAPs such as D-AKAP1
434 (Huang et al., 1997b) and D-AKAP2 (Huang et al., 1997a) have been shown to bind RI as well,
435 thereby making them dual-specific. Recently, other AKAPs such as smAKAP or GPR161 were
19 436 shown to be highly specific and selective for RI subunits (Bachmann et al., 2016; Burgers et al.,
437 2012). The first RIβ specific AKAP, which is the only neuronal binding partner for RIβ
438 identified so far, is the neurofibromatosis 2 tumor suppressor protein merlin (Gronholm et al.,
439 2003). In this study, we provide a comprehensive comparison between the protein localization of
440 RIβ and RIIβ in several brain regions, and show that RIβ co-localizes with MAP2 at the
441 cerebellum. MAP2 was the first AKAP identified to bind RII subunits and tethers PKA with
442 microtubules (Theurkauf and Vallee, 1982). It is only in recent years that we appreciate that
443 some AKAPs can be dual-specific. Based on sequence alignment as well as structural alignment,
444 it can be deduced that MAP2 is an AKAP that satisfies the requirement of binding both RI and
445 RII subunits (Sarma et al., 2010). We thus suggest that preferential binding and interactions of
446 either RI or RII with a potential dual specific AKAP is likely to be cell specific and may be
447 determined by the predominant R subunit that is expressed in a certain location.
448 Little is known about AKAPs that assemble and integrate RIβ holoenzyme signaling at
449 cellular and subcellular regions; however, recent discoveries suggest that RIβ
450 compartmentalization can indeed be linked to neurodegenerative disease. A point mutation in the
451 dimerization and docking (D/D) domain of human RIβ, for example, has been associated with a
452 new type of a familial neurodegenerative disease presenting with dementia and Parkinsonism
453 (Wong et al., 2014). These patients also show an increase of RIβ in neuronal inclusions. This
454 mutation can thus be classified as a targeting defect that can be validated in mouse models and in
455 patient samples where we anticipate that RIβ localization will be altered. Although the
456 pathophysiological mechanism by which this mutation leads to neurodegenerative disease
457 remains to be investigated, this point mutation further emphasizes the importance of precisely
458 controlled PKA isoform localization. Our mosaic maps that can be downloaded and queried in
20 459 great detail represent the endogenous expression and localization of PKA isoforms. These
460 images lay the foundation for studying biological mechanisms of neurodegenerative diseases at
461 subcellular and molecular levels where PKA regulation may be dysfunctional both in mouse
462 models and patient samples.
463
464
465
466
467
468
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475
476
21 477 MATERIAL AND METHODS
478 mKO2 Plasmids
479 The mKO2-tagged PKA regulatory (R) subunits were generated by fusing respective R subunits
480 with mKO2 to the C terminus with PCR and inserted between EcoRI and NotI sites in pcDNA3
481 (Invitrogen). There is a SalI site as linker in RIα, RIβ, and RIIβ constructs and a BamHI site as
482 linker in the RIIα construct.
483 Antibody information
484 Primary antibodies: Rabbit monoclonal anti-PKA R2B Abcam catalog # AB75993 (RRID:
485 AB_1524201). Dilution 1:100. The same pattern of staining was obtained when using Anti- PKA
486 RIIβ mouse monoclonal BD Biosciences Cat #610625 (RRID: AB_397957) at 1:200 dilution for
487 immunofluorescence. Anti-PKA RIα Abcam catalog# ab60064 (RRID: AB_2168081) dilution
488 1:1000 for WB. Anti- PKA RIIα Abcam catalog number # ab38949 (RRID: AB_725890)
489 dilution 1:1000 for WB. Mouse anti-MAP2 Sigma Aldrich M-4403 antibody at 1:200 dilution.
490 Secondary antibodies: Cy3-Donkey anti sheep (Jackson ImmunoResearch Laboratories) (RRID:
491 AB_2315778), Alexa Fluor 488 Donkey anti rabbit (Jackson ImmunoResearch Laboratories)
492 (RRID: AB 2313584), Alexa 568 Fluor Donkey anti mouse (Jackson ImmunoResearch
493 Laboratories). All secondary antibodies were used at 1:250 dilution. pCREB cell signaling
494 (RRID: AB_2085876). BIII-tubulin Covance Research Products Inc Cat# MMS-435P (RRID:
495 AB_2313773).
496 RIβ antibody purification
22 497 Sheep anti-RIβ polyclonal. R&D systems. Catalog # AF4177 (RRID: AB_2284184) was further
498 purified as described below to eliminate cross reactivity with RIα and used at 1:400 dilution.
499 cAMP-sepharose resin was equilibrated with a washing buffer (20mM MES pH 6.5, 100mM
500 NaCl, 2mM EDTA, 2mM EGTA, 5mM DTT). 2µg purified RIα protein was added to the resin
501 and incubated overnight at 4°C. The next day, access RIα protein was washed from the resin
502 using the washing buffer. Resuspended lyophilized RIβ antibody was added to the resin and
503 incubated overnight at 4°C. The following day, resin was centrifuge 3000rpm for 5min at 4°C.
504 Supernatant together with 3 resin washes with TBST were collected and concentrated using
505 Amicon centrifugal concentrator. Final antibody concentration was 0.5mg/ml.
506 Dot Blot experiment
507 Purified proteins were spotted on a nitrocellulose membrane. The membrane was incubated at
508 room temperature for ½ hr to ensure that the blots are dry. Membrane was blocked with 5% dry
509 milk in TTBS (50mM Tris, 0.5M NaCl, 0.05% Tween-20, pH7.4) for 1 hr at room temperature.
510 Membrane was incubated with primary antibodies for 1hr at room temperature. Following 3X
511 washes with TTBS membrane was incubated with secondary antibodies conjugated with HRP for
512 30min. Following 3X washes with TTBS membrane was incubated with ECL reagent.
513 Cultured cells and immunostaining
514 10T1/2 and 293T cell lines, received from research labs, tested negative for mycoplasma
515 contamination based on DAPI staining. 10T1/2 cell line was not found to be on the list of
516 commonly misidentified cell lines (International Cell Line Authentication Committee). HEK
517 cells were found to be on the list of commonly misidentified cell lines. The authors performed
518 no further authentication of the cell lines.
23 519
520 To validate antibody specificity, 10T1/2 cells maintained in DMEM supplemented with 10%
521 fetal bovine serum (FBS) and 2mM GlutaMAX were grown to 80% confluency on glass
522 coverslips in a 37°C incubator with 10% CO2. Transient transfections were carried out using
523 2mL of Lipofectamine reagent (Invitrogen) and 0.5 mg of DNA encoding RIβ-MKO2, RIα-
524 MKO2, RIIβ-MKO2 and RIIα-MKO2 plasmids. Approximately 20 h after transfection, cells
525 were fixed with 4% paraformaldehyde. For immunostaining, cells were permeabilized with 0.3%
526 Triton X-100 and blocked with 1% normal donkey serum, 0.5% BSA, and 50 mM glycine. Cells
527 were stained with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room
528 temperature. Coverslips were mounted on glass slides using ProLong Gold antifade reagent with
529 Dapi (Invitrogen). Images were acquired using FluoView 1000 confocal laser scanning
530 microscope on a 60x objective lens with an NA of 1.45 (Olympus).
531 Primary cultured cells
532 Hippocampal and cortical neurons were dissected from embryonic day 18 (E18) Sprague Dawley
533 rats, and dissociated with papain. Cells were transfected by Amaxa electroporation (Lonza),
534 cultured in Neurobasal medium with B27 supplemen, 2 mM GlutaMAX, 50 U/ml penicillin and
535 50 µg/ml streptomycin as previously described (Lin et al., 2008). (Approved Animal Protocol:
536 S03182R).
537 Subjects for mouse brain mosaic images
538 All experiments involving vertebrate animals conform to the National Institute of Health Guide
539 for the Care and Use of Laboratory Animals (NIH publication 865-23, Bethesda, MD, USA) and
540 were approved by the Institutional Animal Care and Use Committee (IACUC) of the University
24 541 of California San Diego (Approved Animal Protocol: S03172m). In these experiments we used
542 male C57BL/6 mice approximately two months old.
543 Tissue preparation and processing
544 Mice were fully anesthetized and PBS was perfused transcardially for 3 min followed by 4%
545 paraformaldehyde for 10 min. The brain was removed and post-fixed in 4% paraformaldehyde
546 overnight at 4°C. This overnight fixation step ensures better quality of vibratomed sections and
547 reduces the number of tears in the final sections.
548 Sagittal cerebellum sections or coronal sections were cut on a Leica vibratome at a thickness of
549 75-100 microns. If not processed at the same day, tissues were stored at −20°C in cryoprotectant
550 solution (30% glycerol, 30% ethylene glycol in PBS) until processed.
551 Next, free-floating sections were washed three times with 1×PBS for 5 min. Sections were then
552 blocked with 3% normal donkey serum, 1% bovine serum albumin, 1% fish gelatin, 0.1% Triton
553 X100, and 50mM glycine in PBS for 1h at room temperature. Primary antibodies were applied
554 overnight at 4°C. The following day, sections were washed three times with 1×PBS for 5 min
555 and then stained with the secondary antibody for 2 hours at room temperature. Sections were
556 washed three times with 1×PBS for 5 min before mounting on glass slides using ProLong Gold
557 antifade reagent with Dapi (Invitrogen).
558 Specimen preparation and imaging
559 Because wide-field brain mosaics were acquired at close to the resolution limit of light
560 microscopy, good structural preservation, and tissue quality were essential. To avoid any
561 structural degradation associated with freezing, fixed tissue was processed unfrozen and
25 562 sectioned on a vibrating microtome. Only sections devoided of tears, fold, and other defects were
563 chosen for analysis. Care was taken during mounting and fluorescent labeling to minimize
564 compression and optimize staining and imaging conditions.
565 Wide scale data acquisition
566 A FluoView 1000 (Olympus Center Valley, PA, USA) equipped with 20x, 40x NA 1.3 oil or 60x
567 NA 1.45 oil immersion objective and a high-precision motorized stage was used to collect the
568 large-scale 3D mosaics of each tissue section. The boundaries (in x, y, and z) of the tissue section
569 were defined using the Multi-Area Time Lapse function of ASW 1.7c microscope operating-
570 software provided by Olympus (Olympus, Center Valley, PA, USA). The software automatically
571 generates a list of 3D stage positions covering the volume of interest, which are computed using
572 the dimensions of a single image in microns, degree of overlap between adjacent images and z-
573 step size. Individual image tiles were 1024 × 1024 with a pixel dimension of 0.62 μm; overlap
574 between two adjacent images (x–y) was 10% and the z-step was ~0.5 mm/section; there is no
575 overlap in z. The specimen was excited sequentially with a laser at two different wavelengths:
576 488 nm and 561nm. The final data is stored in a RGB image volume, where the color channels
577 map the specimen susceptibility at wavelengths 561 and 488 nm. Unprocessed data was stored as
578 a single image stack containing information about the relative position of each image tile.
579 Image processing
580 The tiles were stitched together post-data acquisition to generate the final reconstructed 2D
581 volume using National Center for Microscopy and Imaging Research (NCMIR) developed
582 ImageJ Mosaic Plug-ins (RRID: SCR 001935), flat field, normalize, align, and combine into a
583 mosaic (Berlanga et al., 2011; Chow et al., 2006) Software is available for download (Chow et
26 584 al., 2006). The resulting reconstructed mosaic image was downloaded and opened in Adobe
585 Photoshop CS2 (Adobe Systems Inc., San Jose, CA, USA) (RRID: SCR 002078) to rotate, crop,
586 and adjust color balance of the image.
587 Image deposition into the cell centered database
588 Mosaic images were acquired using an automated imaging workflow system developed at
589 National Center for Microscopy and Imaging Research (NCMIR) (RRID: SCR 002655) to
590 upload imaging data imaging data with its associated metadata directly from the microscopes in
591 our facility and register them as microscopy products within the Cell Centered Database
592 (CCDB). This complex system for data collection, storage, and manipulation funnels large
593 numbers of valuable datasets directly into the CCDB (RRID: SCR 002168) (Martone et al.,
594 2008). Three datasets resulting from this publication will be released to the public through the
595 CCDB. Researchers will be able to browse or download our large-scale mosaics through the
596 CCDB at http://www.cellimagelibrary.org/images?k=project_20403&simple_search=Search.
597 Because these data sets are quite large and rich in information, each of these reconstructed
598 mosaic images will be viewable at full resolution and annotatable without downloading using the
599 Web Image Browser (WIB) (RRID: SCR 007015), a visualization program based on GIS
600 technology similar to that used for Google maps, but specialized for microscopy imaging data.
601 Through the WIB, users can turn on and off channels and adjust contrast of large microscopic
602 imaging data sets.
603 Electron microscopy
604 Primary hippocampal cortical cells were transfected with miniSOG-tagged RIβ using Amaxa
605 electroporation (Lonza). Samples were treated as described previously in details (Perkins, 2014).
27 606
607 Validation of shRNA lenti-viruses knockdown efficiency
608 293T cells were co-transfected with either PRKARIβ or PRKARIIβ rat cDNA (Origene) and
609 with scrambled shRNA (Sigma) or shRNAs targeting PRKARIβ (5’-
610 CGGCAGAAGTCAAACTCACAGTGTGATTC-3’, purchased from Origene) or PRKARIIβ (#
611 1-5’-CGTCATCGACAGAGGAACATT-3’; # 2-5’-CGATGCAGAGTCCAGGATAAT-3’ and #
612 3-5’-CCTTCAGGAGAATAATAGTAA-3’). shRNA hairpins were cloned into an NheI site of
613 the p156RRLsin lenti-vector expressing GFP (Dull et al., 1998). 293T cells growing on 60-mm
614 Petri dishes were transfected with 0.5μg plasmids encoding either RIβ or RIIβ cDNA, together
615 with 5μg shRNA against either RIβ or RIIβ, or 5μg negative control. Transfections were
616 performed using Lipofectamin TM 2000 (Invitrogen) according to manufactures’ instructions.
617 Protein lysates prepared 48 hours post-transfection using RIPA buffer supplemented with
618 protease inhibitors (Roche). Western blot analyses carried out using RIβ or RIIβ antibodies as
619 well as actin antibody for loading control were used. Lentiviral particles were then prepared as
620 previously described (Ikawa et al., 2003).
621 Down-regulation of RIβ/ RIIβ in neurons and detection of CREB phosphorylation
622 Primary hippocampal neurons were isolated as described above or obtained from (Life
623 TechnologiesTM: A10840-01). Cells were plated in Neurobasal media supplemented with 10%
624 FBS, 1% glutamine, 2% G21, and Pen/strep. The following day, media is changed to serum free
625 Neurobasal. On day 6-7, cells were transduced with the indicated lentivirus (1x10e9/ml) at
626 1:1000 dilution overnight, and media replaced the next day. After allowing an additional day of
627 recovery, cells are then pulsed for 30 minutes with 100 µM forskolin (Sigma # F6886) prior to
28 628 fixtation via a 15 minute pre-fix with 4% PFA in PBS added at a 1:1 ratio to the existing media,
629 followed by 10 minutes in 4% PFA solution only. Cells were then probed for p-CREB (cell
630 signaling) BIII-tubulin (BioLegendTM MMS-435P) and the corresponding fluorescent secondary
631 antibodies as indicated, as well as DAPI. Cells were then imaged via fluorescent microscopy,
632 and cell total corrected fluorescence of p-CREB in infected neurons was obtained using ImageJ.
633 Statistical analyses were performed using Tukey’s multiple comparisons test under one-way-
634 ANOVA (GraphPad Prism6 software). Multiplicity adjusted P-values are reported.
635 Acknowledgments
636 We thank Dr. Maryann Martone from the national center for microscopy and imaging research
637 (NCMIR) for valuable discussions and critical comments on the manuscript. We thank M.H.E
638 laboratory members at NCMIR for helpful discussion, especially Dr. Tom Deerinck and Dr.
639 Angela Cone (NCMIR). We thank Hiro Hakozaki (NCMIR) for technical support. We thank Dr.
640 Harvey Karten, UCSD for valuable discussions on the manuscript. We are particular grateful to
641 Dr. Davide Dulcis for critical comments on the manuscript. We thank all members of the S.S.T.
642 especially: Dr. Yuliang Ma for plasmids and valuable discussions, and Dr. Kristofer J Haushalter
643 for antibody specificity immunoblots. We thank Dr. Ayelet Gonen for helping with statistical
644 analysis. This work was supported by National Institute of Health Grants DK054441 (to S.S.T).
645 This work was supported by grants from the NIH National Institute of General Medicine
646 Sciences under award number P41GM103412 to Mark Ellisman, operating the National Center
647 for Mark H. Ellisman, operating the National Center for Microscopy and Imaging Research, and
648 through award number 2R01 GM082949 to Mark H. Ellisman operating the Cell Image
649 Library/Cell Centered Database.
29 650
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789 FIGURE LEGENDS
790 Figure 1. Overview of the regional distribution of RIβ and RIIβ across brain regions. Wide-
791 field views of a high-resolution large-scale mosaic image for a full coronal tissue slice are shown
792 for RIβ (A/red in colored image) and RIIβ (B/green in colored image). The tissue section was
793 labeled with anti-RIβ and anti-RIIβ specific antibodies. The mosaic image was made up of 105
794 tiles. Each tile is a maximum intensity projection of a stack of 15 Z-sections that were stitched
795 together to reconstruct this single, high resolution 2D image. Colored image: (A) Scale bar inside
33 796 the full mosaic image: 1mm. Scale bar inside the small box: 100µm. (B) Full resolution sample
797 of the mosaic allows examination at higher magnification. White boxes represent the areas from
798 which the (B) was captured. DG, Dentate Gyrus. Scale bar: 10 µM. Abbreviations: CPu, Caudate
799 putamen. Rh, rhomboid thalamic nucleus. Re, reuniens thalamic nu. Rt, reticular thalamic
800 nucleus. Arc, arculate nucleus. Pir, piriform cortex. DEn, dorsal endopiriform nucleus. SubI,
801 subincertal nucleus. RSG, retrosplenial granular cortex. MHb, medial habenular nuclear. RSV,
802 retrosplenial granular. MF, mossy fibers.
803 Figure 1-figure supplement 1. Antibody specificity. Left panel: Commassie blue stained gel
804 or western blot of purified RIα/RIβ proteins detected by RIα/RIβ antibodies. Right panel: Dot
805 blot analysis. Each row of the nitrocellulose membrane was spotted with all four purified PKA
806 regulatory subunits as indicated and followed by specific antibody detection. Lower panel:
807 10T1/2 mouse cell culture were transfected with RIβ-MKO2 (A) or RIα-MKO2 (B) and were
808 immunostained with RIβ antibody (A2, B2). Merge images are shown (A3, B3). No unspecific
809 staining was detected in controls for secondary antibodies alone (A4-D4). Pre-absorption of RIβ
810 or RIIβ antibodies with purified RIβ or RIIβ proteins respectively resulted in blocking the
811 antibody signal (A5,C5).
812 Similar experiment was done with overexpressing RIIβ-MKO2 (C1) or RIIα-MKO2 (D1). Cells
813 were immunostained with RIIβ antibody (C2, D2). Merge images are shown in (C3.D3). No
814 unspecific staining was detected in controls for secondary antibodies alone (C4, D4).
815 Figure 1-figure supplement 2. Specificity of secondary antibodies. Wide- field views of a
816 high-resolution large-scale mosaic image for a full coronal tissue slice are shown for secondary
817 antibodies alone: Alexa Fluor 488 donkey anti rabbit (green) and CY3 Donkey anti sheep (red)
34 818 including dapi staining (blue). (2) Full resolution sample of the mosaic allows examination at
819 antibody controls at higher magnification. No staining was detected. White boxes represent the
820 areas from which (1) and (2) was captured. DG, Dentate Gyrus.
821 Figure 2. RIβ is predominantly localized to cell body and dendrites in different
822 hippocampal cell types. A. Coronal sections were taken from a mouse brain and labeled with
823 anti-RIβ (red) and anti-RIIβ antibodies (green). Left: mosaic image of a full brain section made
824 up of 105 tiles. Scale bar: 1mm. Right: mosaic image of the hippocampus region made up of
825 1413 tiles, ten Z sections using a 60x objective lens. Scale bar: 200µm. B-C. Top: Mosaic
826 image of the hippocampus at reduced resolution with anti RIβ (B) or anti RIIβ (C). The white
827 box represents the area from which the middle image was captured. Middle: Higher resolution
828 sample of the mosaic showing the pyramidal cell layer in CA1-2 subfield. Anti- RIβ (left) or
829 anti-RIIβ (right). The white box represents the area from which the Bottom image was captured.
830 Scale bar: 200µm. Bottom: A full resolution of the mosaic shows a representative interneuron
831 cell within the hippocampus region. Anti-RIβ (left) or anti-RIIβ (right). Scale bars: 10µm.
832 Figure 3. RIIβ is the predominant isoform in cell axons across various hippocampal
833 subfields. A. A full representative mosaic of coronal section through the dorsal hippocampus at
834 reduced resolution. An overview of RIβ (red), RIIβ (green) and RIβ/RIIβ immunostaining at
835 various subfields. The mosaic image made up of 1413 tiles, ten Z sections using a 60x objective
836 lens. The white box represents the area from which B-E subfield full resolution images were
837 captured. B. Hilus of dentate gyrus. C. Mossy fiber of the CA3. D. Stratum lacunosum-
838 moleculare. E. Alveus. Abbreviations: CA1 stratum oriens; p, stratum pyramidale; r, stratum
839 radiatum; lm, stratum lacunosum-moleculare; m, dentate molecular layer (stratum moleculare);
840 g, granule cell layer (stratum granulosum); h, hilus proper. Scale bar: 25µm.
35 841 Figure 3-figure supplement 1. Mosaic images of the hippocampus allow tracing RIIβ along
842 an axonal pathway and visualizing axonal boutons in the full resolution mosaic image.
843 Middle image: Immunostaining of RIIβ in full representative mosaic of coronal section through
844 the dorsal hippocampus. Subfields of the hippocampus are marked as in the main figure 3.
845 Numbers in white boxes represents the area from which 1-6 full resolution images were
846 captured. 5 and 6 show full resolution axonal pathway that emerges from the granule cells of the
847 dentate gyrus and pass through the hilus to the mossy fibers at the CA3. 2,3,4 show a full
848 resolution image of RIIβ staining the axonal terminal boutons.
849 Figure 4. RIβ is the predominant isoform at the cerebellum. A. An overview of a full
850 representative sagital section of one folium of the cerebellum immunostained with anti-RIβ (A)
851 or anti- RIIβ (A’) at reduced resolution. The mosaic image is made up of 120 tiles, 21 Z sections
852 using a 40x objective lens. Scale bar: 100 µm B. Same as A. RIβ (red), RIIβ (green) and Dapi
853 (blue). White boxes represent the areas from which C-D images were captured. C.Cerebellar
854 Purkinje cells at the molecular layer (m) and the granular layer (g) are indicated. Right side: full
855 resolution views of Purkinje cells. RIβ is localized to the somata and the dendrites of these cells.
856 RIIβ is less abundant at their dendrites. Scale bar: 25µm. D. Full resolution views taken from the
857 granular cell layer (g) and the white matter.
858 Figure 5. RIβ co-localizes with MAP2. Sagital sections of the cerebellum were co-stained with
859 microtubule-binding protein (MAP2) antibody, a dendritic marker, and RIβ antibody (A) or RIIβ
860 antibody (B). Primary cortical hippocampal cultured cells were co-stained with MAP2 antibody
861 and RIβ antibody (C) or RIIβ antibody (D).Dapi staining for the nucleus is shown in blue. Scale
862 bars: 20µm.
36 863 Figure 6. Nuclear localization of RIβ subunit across various brain regions. Full resolution
864 views of different brain regions were taken from the mosaic maps or primary cell staining to
865 show the nuclear co-localization. RIβ (red), RIIβ (green) and dapi (blue). One slice of the Z stack
866 is shown A. Purkinje cells of the cerebellum. B. Interneurons in the hippocampus. C. Cells from
867 the thalamus. D. Cells from the hypothalamus. E. Primary cortical/hippocampal cells. Scale bar:
868 10µm.
869 Figure 6-figure supplement 1. Tissue staining with secondary antibodies alone shown as
870 negative controls for brain regions indicated in main Figure 6. Hippocampus in wide field
871 views stained with Alexa Fluor 488 donkey anti rabbit (green) and CY3 Donkey anti sheep (red)
872 including dapi staining (blue). Numbers represent the areas from which higher magnifications
873 were taken. Interneuron (1) and neurons from CA1 region (2). Hypothalamus in wide field view
874 and at higher magnification (3). No unspecific staining from secondary antibodies was detected.
875 Figure 7. miniSOG-tagged RIβ localizes RIβ to the mitochondria and to the nucleus using
876 electron microscopy. Primary hippocampal/cortical cells were electrophorated with miniSOG
877 tagged RIβ. Mersalyl acid was used in the blocking step to reduce background nonspecific
878 labeling of mitochondrion. The differential contrast generated between non-transfected nucleus
879 (A) and a transfected nucleus (B) following photooxidation is evident. RIβ is localized to the
880 nucleus and nuclear envelope. The differential contrast generated between non-transfected
881 photo-oxidized mitochondrion (C) and a transfected photo-oxidized mitochondrion (D) is
882 evident. The well-preserved mitochondrion allows detecting RIβ at the mitochondrial cristae and
883 the inner membrane. 126 transfected darkened mitochondria and 71 non-transfected light
884 mitochondria were counted. A representative image for non-transfected (C) and transfected (D)
885 is shown.
37 886 Figure 8. Down-regulation of RIβ in neurons reduces CREB phosphorylation. A. Primary
887 hippocampal cells were treated with shRNAs against RIβ, RIIβ or irrelevant control. CREB
888 phosphorylation was induced by forskolin incubation. Intensity of CREB phosphorylation at
889 Ser133 was quantified in nucleus (DAPI) of neuronal cells (βIII tubulin) that were infected by
890 the GFP-shRNA lentiviruses (representative cells are indicated by arrows). Either non-neuronal
891 GFP expressing cells or neuronal cells that did not express GFP were excluded from the analysis
892 (examples are indicted by a circle). Representative infected neurons depicting p-CREB, dapi,
893 βIII-tubulin as well as GFP (in squares) are shown in a close up. B. Relative fluorescence of
894 pCREB was quantified for each condition using imageJ. Statistical analyses were performed
895 using Tukey’s multiple comparisons test under one-way-ANOVA (GraphPad Prism6 software).
896 Adjusted P-values are reported. In the RIβ knockdown there is a significant reduction in pCREB
897 fluorescence intensity when compared to control (****p<0.0001) or to RIIβ (***P=0.0003). No
898 statistical significance between control and RIIβ (p=0.3999). Results were assessed from three
899 independent experiments. ~50 cells were quantified for each condition.
900 Figure 8- figure supplement 1. Validation of shRNA knockdown efficiency. Western blot
901 analysis showing silencing of RIβ (A) and RIIβ (B). Native 293T cells (-) were transfected with
902 either RIβ (+) or RIIβ cDNA (+). 293T cells in lane 3-5 (A) and lane 3-6 (B) were co-transfected
903 with either RIβ cDNA (A) or RIIβ cDNA (B) together with a scrambled shRNA (shRNA IRR) or
904 shRNA constructs targeting RIβ (A) or RIIβ (B). shRNA RIβ (Origene) and shRNA RIβ (lenti)
905 differ in their lentiviral backbone, the later one also expresses GFP. All the shRNA RIIβ
906 construct were cloned into the lentiviral vector expressing GFP, and based on the results we
907 continue working with shRNA RIIβ clone #2. Actin was used as a loading control. C.
908 Immunofluorescence signal reduced by shRNAs against RIβ or RIIβ. Representative primary
38 909 neuronal cells infected with either shRNA targeting RIβ/RIIβ or a scrambled negative control
910 (shRNA SCR). GFP expression from the shRNA vector marks infected cells. Cells expressing
911 shRNA-targeting RIβ/RIIβ display a significant reduction in fluorescence signal intensity of
912 RIβ/RIIβ antibody as compared to cells expressing shRNA SCR. CY5 conjugated secondary
913 antibodies were used for both RIβ and RIIβ primary antibodies D. Corrected total cell
914 fluorescence was measured for each condition using imageJ. Mean fluorescence of background
915 readings in area of selected cell were omitted from integrated density. The level of GFP
916 fluorescence was normalized to 1 for each condition. ~30 cells were quantified for each
917 condition. P value <0.0001, Student’s t-test.
918
919
39 RIβ RIIβ
Figure 1 RIβ an body RIα an body
Figure 1- figure supplement 1 Controls of secondary an bodies only: Alexa Fluor 488 Donkey an rabbit Cy3 Donkey an sheep Dapi
1
2
1
2
Figure 1- figure supplement 2
A RIβ RIIβ RIβ RIIβ a o E p CA2 r D CA1 lm C m MF DG CA3 B h
B Hilus of the Dentate Gyrus
RIβ DAPI RIIβ DAPI RIβ RIIβ DAPI C Mossy Fibers of the CA3
RIβ DAPI RIIβ DAPI RIβ RIIβ DAPI D Stratum lacunosum-moleculare
RIβ DAPI RIIβ DAPI RIβ RIIβ DAPI Alveus E
RIβ DAPI RIIβ DAPI RIβ RIIβ DAPI
Figure 3 Stratum Radiatum Alveus Mossy Fibers
1
a 1 o
P CA2 CA1 6 2 3 r
2 4 lm CA3
Stratum Radiatum m MF
DG 6 h 5
Stratum lacunosum- moleculare Hilus of the dentate gyrus
3 4 5 Figure 3- figure supplement 1
Figure 3 Figure 3 A RIβ A’ RIIβ
Cerebellum RIβ DAPI RIIβ DAPI RIβ RIIβ DAPI B B’ B B’
C C’
D D’
C C’ C C’
m g D D’ D D’
g Figure 4 A RIβ MAP2 RIβ MAP2
B RIIβ MAP2 RIIβ MAP2
C RIβ MAP2 RIβ MAP2
D RIIβ MAP2 RIIβ MAP2
Figure 5
Controls of secondary an bodies only: Alexa Fluor 488 Donkey an rabbit Cy3 Donkey an sheep Dapi
Hippocampus 1 Interneurons
1 2
Hypothalamus 2 CA1 3 3
Figure 6 –figure supplement 1
A. B.
p"CREB' DAPI' B3'Tubulin' GFP' Merged' SCR' 'KD' β І R 'KD' β I І R
Figure'8'
Figure 8 A. B.
(-) (+) (-) (+) RIIβ RIβ Ac n Ac n
C. D. shRNA RIβ shRNA SCR **** ****
GFP RIβ an body GFP RIβ an body shRNA RIIβ shRNA SCR
GFP RIβ GFP RIIβ GFP RIβ RIIβ Corrected total cell fluorescence shRNA RIβ shRNA RIIβ shRNA SCR GFP RIIβ an body GFP RIIβ an body
Figure 8-figure supplement 1