Neurons are highly complex, compartmentalized cells that respond to a wide range of physiological and pathological signals by regulated changes to their transcriptome and proteome. A critical component in this process is the synthesis of new proteins, both in the cell body and at the synapse (Davis and Squire, 1984; Hafner et al., 2019; Li and Götz, 2017; Schanzenbächer et al., 2016). New protein synthesis is required for the formation, retrieval, and updating of long-term memories (LTMs), distinguishing this process from short-term memory (Costa-Mattioli et al., 2009; Jarome and Helmstetter, 2014; Lopez et al., 2015). One form of LTM is spatial LTM, which is used by organisms to recall spatial information about the environment, and is formed through the process of spatial LTM consolidation (Squire et al., 2015). During this process, neurons undergo a series of changes in different brain regions, with the hippocampus being identified as one of the most important brain structures in both spatial learning and LTM (Clopath, 2012; Mayford et al., 2012; Ziegler et al., 2015). Furthermore, inhibition of protein synthesis in the hippocampus, especially within the first 24 hr following spatial learning, has been demonstrated to prevent the formation of spatial LTM (Alkon et al., 2005; Inda et al., 2005; Jarome and Helmstetter, 2014). Therefore, identifying precisely which proteins need to be newly synthesised during spatial LTM consolidation is crucial in dissecting the underlying molecular mechanisms.

Several proteins and pathways have been linked to spatial LTM formation, as shown by candidate-based studies in which rodents were subjected to behavioural paradigms, such as the active place avoidance (APA) or Morris water maze test in order to induce spatial LTM formation (Merlo et al., 2015; Paul et al., 2009; Plath et al., 2006). However, a non-biased proteomic analysis of de novo protein synthesis during the formation of spatial LTM has not been previously achievable, because de novo synthesised proteins are chemically indistinguishable from those that are already present in the cell. This limitation can, however, be overcome through non-canonical amino acid (NCAA) labelling of newly synthesised proteins. In this technique, NCAAs can be administered for a given period, during which they are integrated into the nascent polypeptide chain (Figure 1A) (Hinz et al., 2013). Unlike most other de novo protein tagging techniques, NCAA incorporation enables newly synthesised proteins to be visualised using fluorescent non-canonical amino acid tagging (FUNCAT), or to be purified using bio-orthogonal non-canonical amino acid tagging (BONCAT) (Figure 1B) (Hinz et al., 2013). This is achieved by reacting the azide group of the NCAA with a dibenzocyclooctyne (DIBO)-bearing tag, using strain-promoted azide-alkyne cycloaddition (Figure 1B) (Beatty et al., 2010).

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WT 30 minute APA behavioural data.

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Two widely used NCAAs are the methionine surrogates azidohomoalanine (AHA) and azidonorleucine (ANL). In AHA labelling, the endogenous translational machinery is used to tag newly synthesised proteins with AHA (Figure 1A) (Dieterich et al., 2006; Hinz et al., 2013; Ullrich et al., 2014). ANL, on the other hand, allows for cell-type-specific NCAA labelling, as it is not recognized by the eukaryotic methionine tRNA synthetase and therefore does not natively integrate into de novo synthesised proteins (Link et al., 2006). ANL instead requires the presence of a mutant tRNA synthetase, such as NLL-MetRS, for its incorporation (Figure 2A) (Ngo et al., 2013). Genetically restricting NLL-MetRS expression enables cell-type- or tissue-specific incorporation of ANL. Both AHA and ANL labelling have been used in a wide range of cell-types, tissues and organisms (Alvarez-Castelao et al., 2019; Alvarez-Castelao et al., 2017; Erdmann et al., 2015; Liang et al., 2014; Lopez et al., 2015; McClatchy et al., 2015; Ullrich et al., 2014). However, even though AHA and ANL labelling display an enhanced versatility compared to other de novo protein labelling techniques, such as stable isotype labelling with amino acids in cell culture (SILAC) or puromycin labelling, they are yet to be more widely used to examine the de novo proteomic changes which occur during complex rodent behaviour.

Figure 2

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Here, we detail the use of NCAA labelling to identify changes in de novo protein synthesis which occur during the formation of spatial LTM in mice. For this, we generated the RC3 mouse strain, which enables Cre recombinase-dependent targeting of NLL-MetRS expression for cell-type- or tissue-specific incorporation of ANL. Upon crossing the RC3 strain with Camk2a-Cre mice, we successfully restricted ANL labelling of newly synthesised proteins to hippocampal neurons. We combined this labelling technique with SWATH-MS (sequential window acquisition of all theoretical fragment ions mass spectrometry) to examine how the hippocampal de novo proteome is altered in mice following spatial LTM formation. Our results confirmed a number of previously established memory-related proteins to be altered in synthesis during spatial LTM formation, and also highlight potential novel memory mechanisms, such as alterations in mRNA splicing.

In our study, we used NCAA labelling in combination with FUNCAT, BONCAT and SWATH quantitative proteomics to examine how de novo protein synthesis is altered in the initial stages of spatial LTM consolidation. Using FUNCAT labelling with the non-canonical amino acid AHA, we first observed that as expected, protein synthesis was increased in the hippocampus of mice during spatial LTM formation (Jarome and Helmstetter, 2014). We further refined our approach by generating the RC3 mouse strain, which, upon crossing with the Camk2a-Cre strain and administration of ANL, allowed for neuron-specific labelling of de novo synthesised proteins specifically in the hippocampus. Using BONCAT in combination with SWATH-MS quantitative de novo proteomics, we identified a total of 1782 proteins which were newly synthesised in hippocampal neurons, 156 of which were found to be significantly altered in synthesis following spatial LTM formation, with BONCAT-WB being used to independently validate a subset of these proteins. These regulated proteins are involved in a wide range of cellular functions, including mRNA splicing, ATP hydrolysis-coupled proton transport, vesicle-mediated transport, biogenesis of mitochondrial complex I, and signalling through Rho GTPases. Our de novo proteomic data suggest a role for these proteins, and the pathways they are involved in, in spatial LTM formation and, more generally, demonstrate that NCAA labelling is a robust, non-biased experimental strategy to examine how the de novo proteome is altered during complex rodent behaviour.

Previously, NCAA labelling has been used in rodents to observe changes in de novo protein synthesis during development (Calve et al., 2016), to compare different cell-types following environmental enrichment (Alvarez-Castelao et al., 2017), and to examine changes which occur in mouse models of disease (Evans et al., 2019; McClatchy et al., 2015). To the best of our knowledge, the current study represents the first use of NCAA labelling to examine spatial LTM formation in mice.

NCAAs, such as AHA and ANL, have typically been delivered to mice through diet, with labelling periods ranging from 6 to 21 days (Alvarez-Castelao et al., 2017; McClatchy et al., 2015). Spatial LTM consolidation is dependent on multiple temporally spaced phases of protein synthesis (Fioriti et al., 2015; Meiri and Rosenblum, 1998; Ozawa et al., 2017), with the first 24 hr following training being the most sensitive to the inhibition of protein synthesis (Freeman et al., 1995; Quevedo et al., 1999). Given that the majority of changes to the hippocampal de novo proteome are thought to occur within this time frame, we considered the longer labelling periods used in previous studies not to be suitable for examining spatial LTM-induced protein synthesis. We therefore delivered AHA and ANL via intraperitoneal injection rather than dietary administration, enabling us to label newly synthesised proteins within a 16 hr time window (Figure 2F). An additional advantage of labelling for this shorter period is the increased temporal specificity, with a higher proportion of tagged proteins being synthesised only in response to the behavioural paradigm studied, compared to those that would be synthesised after a longer period of time.

Using ANL labelling in combination with SWATH-MS, we obtained a similar degree of proteome coverage to the only other study we are aware of that has used ANL labelling in mice (Alvarez-Castelao et al., 2017). However, given that the neuronal proteome is believed to consist of around 6000 proteins (Schanzenbächer et al., 2016), more invasive delivery techniques such as cranially implanted osmotic pumps may be required to achieve a deeper degree of labelling than observed in either study.

In order to investigate protein synthesis specifically during spatial LTM formation, we established an accelerated version of the five-day APA paradigm, which relies upon a singular training event (Figure 1C), making it easier to capture the time-period during which spatial LTM formation occurs. We confirmed that this 30 min APA task induces spatial LTM by examining two defining characteristics of long-term memory; the ability to persist for long periods of time and the requirement of new protein synthesis (Figure 3—figure supplement 1, Figure 3—figure supplement 2) (Costa-Mattioli et al., 2009; Rosenberg et al., 2014).

Mice that are exposed to a novel environments and stimuli could potentially experience increased levels of stress while undertaking behavioural tasks, which may alter protein synthesis (Chandran et al., 2013; Moncada and Viola, 2007). We controlled for this by using paired, yoked controls and confirmed that these non-trained mice showed similar levels of stress as their trained counterparts (Figure 1—figure supplement 2). We therefore conclude that the observed changes in protein synthesis between trained and non-trained mice were likely induced by spatial LTM formation, rather than confounding factors including training and stress.

In our analysis we focused on the hippocampus, as protein synthesis in this region plays a critical role in spatial LTM (Burgess et al., 2002; Jarome and Helmstetter, 2014; Kleinknecht et al., 2012; Merlo et al., 2015). As our initial AHA labelling and FUNCAT analysis confirmed that protein synthesis is altered in the hippocampus during spatial LTM formation (Figure 1E), we chose to use cell-type-specific NCAA labelling in order to restrict de novo protein labelling to hippocampal neurons in vivo.

To restrict the expression of NLL-MetRS to hippocampal neurons, we crossed RC3 mice with the Camk2a-Cre T29-1Stl/J mouse strain. When originally generated, this mouse strain expressed Cre-recombinase only in the CA1 pyramidal neurons of the hippocampus and the testes (Tsien et al., 1996); however, more recent analyses revealed expression in other regions of the hippocampus (Alvarez-Castelao et al., 2017; McGill et al., 2017), possibly reflecting a genetic drift. Our results are consistent with these more recent studies, with RC3xCamk2a-Cre mice expressing NLL-MetRS-EGFP in the neurons of the CA1, CA2, CA3, as well as dentate gyrus regions of the hippocampus (Figure 2D).

Recently, another Cre-dependent mouse strain was established that expresses the mutant tRNA synthetase MetRS-L247G (Alvarez-Castelao et al., 2017). Unlike NLL-MetRS, which is considered to label proteins with ANL only at their amino-terminus (Ngo et al., 2013), MetRS-L247G also replaces internal methionine residues with ANL (Müller et al., 2015; Yang et al., 2018), which likely results in a wider range of protein labelling.

By combining hippocampal neuron-specific ANL labelling with BONCAT-SWATH-MS, we identified 156 proteins which were significantly altered in synthesis during spatial LTM formation induced by the 30 min APA paradigm (Figure 3D). While these changes represent a small proportion of the total-ANL proteome, these findings are in line with previous studies assessing the total proteome in different stages of memory consolidation (Borovok et al., 2016). STRING analysis of the 156 significantly altered proteins revealed that the vast majority of these proteins interact with a least one other significantly altered protein, forming a large, highly interconnected network (Figure 4). This high degree of interconnection would suggest that, as expected, spatial LTM formation induces changes not only in the synthesis of specific proteins but also pathways.

Interestingly, while FUNCAT analysis revealed an overall increase in the amount of new protein synthesis during spatial LTM formation (Figure 3B), our de novo proteomic analysis identified a similar number of proteins with decreased and increased synthesis (68 and 88, respectively) in the 16 hr following training. This would suggest that rather than simply inducing the synthesis of memory-related proteins, spatial LTM memory formation causes neurons to selectively modulate certain pathways and molecular processes.

MCODE analysis of the network-regulated proteins identified five distinct clusters of newly synthesised proteins. These were associated with mRNA splicing, ATP hydrolysis-coupled proton transport, vesicle-mediated transport, biogenesis of mitochondrial complex I, and Rho GTPase effectors. We confirmed the validity of our de novo analysis by examining a number of proteins from these clusters using BONCAT-WB, with all of the examined proteins showing similar changes in synthesis for both detection methods (Figure 5).

Many of the proteins and pathways identified by our analysis have previously been associated with memory formation. Examples are α- and β-adaptin (Ap2b1), which were increased in synthesis during spatial LTM formation (Figure 4 and Figure 5). These two proteins are subunits of the clathrin adaptor protein 2 (AP2) complex, which regulates synaptic levels of GluR1- and GluR2/GluR3-containing AMPA receptors (Garafalo et al., 2015). This process is important in the memory-related processes of long-term potentiation (LTP) and long-term depression (LTD) (Barth and Wheeler, 2008; Hardt et al., 2014), as well as complex rodents behaviours such as visual recognition memory, which is disrupted when the interaction between AP2 and the GluR2 subunit is blocked (Griffiths et al., 2008).

Another memory-related protein identified to be altered in synthesis in our de novo proteomic analysis was the structural subunit A of protein phosphatase 2A, PP2A-A. Both BONCAT-SWATH-MS and BONCAT-WB found that the α isoform of PP2A-A was increased in synthesis during the 16 hr following training (Figure 3D and Figure 5). Inhibition of PP2A has been demonstrated to block LTP (Belmeguenai, 2005), with hippocampal PP2A knock-out animals presenting altered extinction of long-term memory (Wang et al., 2019).

Our de novo proteomic analysis also identified several molecular processes which have yet to be robustly linked to spatial LTM formation. One such process is the regulation of mRNA splicing. We observed that during memory and learning, there was increased synthesis of pre-mRNA-processing factor 31 (Prp31) and β-catenin-like protein 1 (Ctnnbl1), both of which are required for the formation and activation of the spliceosome (Ganesh et al., 2011; Yuan et al., 2005) (Figure 4), while the synthesis of the pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 (DHX15), which is involved in the disassembly of spliceosomes, was decreased (Wen et al., 2008) (Figure 4). We further observed decreased synthesis of serine/arginine-rich splicing factor 6 (SRSF6), which inhibits the splicing of exon 10 of the microtubule associated protein tau (Yin et al., 2012). Taken together, these results suggest that during spatial LTM formation, there may be an alteration the activity of spliceosomes, leading to altered splicing of certain mRNAs such as MAPT, although further examination will be required to confirm if these molecular process are altered in spatial LTM.

The current working model of spatial LTM formation suggests that following spatial training, hippocampal neurons undergo protein synthesis in response to certain stimuli (Kandel et al., 2014; Squire et al., 2015). In our study, we used cell-type-specific NCAA labelling, in combination with a novel 30 min APA protocol and quantitative SWATH-MS de novo proteomics, to examine how the hippocampal de novo proteome is changed during spatial LTM formation. We found that in hippocampal neurons, there was altered synthesis of specific sets of proteins associated with a diverse, yet interconnected set of neuronal pathways and cellular processes following spatial training. In addition to observing altered synthesis of a number proteins already associated with memory, such as α-adaptin, β-adaptin and PP2A-A, we also identified alterations in mRNA splicing as a potential neuronal mechanism which underpins spatial LTM formation. More generally, our findings highlight the potential of cell-type specific NCAA labelling using transgenic mouse strains such as the RC3 mice as a robust tool for identifying and characterising cell-type-specific changes in de novo protein synthesis that occur in response to a wide range of both physiological and pathological stimuli, including complex rodent behaviours.

All data in this study is presented in the included manuscript, supporting files, and source data files. The proteomics data set has been uploaded to the PRIDE repository with the dataset identifier PXD015820.

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Author details

1. Harrison Tudor Evans

   Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Conceptualization, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0322-0554

2. Liviu-Gabriel Bodea

   Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Conceptualization, Supervision, Funding acquisition

   For correspondence

   l.bodea@uq.edu.au

   Competing interests

   No competing interests declared

3. Jürgen Götz

   Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Conceptualization, Supervision, Funding acquisition

   For correspondence

   j.goetz@uq.edu.au

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8501-7896

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