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Identification and Analysis of Synapse-Organizing Complexes in Addiction-Relevant Brain Regions

Thomas Biederer, Yale University

Overview: Aberrations in neuronal connectivity in addiction-relevant brain regions are correlated with drug seeking and relapse. Trans-synaptic adhesion molecules mediate synapse formation and differentiation, including in brain regions relevant for addiction, and we have previously reported that a mouse model lacking a synaptogenic adhesion protein exhibits altered addiction-relevant behaviors (Giza et al, 2013). This agrees with human genome-wide association studies that support that genetic changes in genes encoding synapse-organizing adhesion proteins can increase the risk for addictive behaviors (Muskiewicz et al., 2018).

These genetic studies need to be complemented by proteomic analysis of synapse-organizing pathways in addiction-relevant brain regions so that functional studies of these molecules can be developed. To aid in these efforts, Dr. Biederer has led during the past funding period proteomic studies of synaptic composition and oversaw a Pilot Project Grant, “Mapping the Proteome of the Synaptic Cleft through Reporter Proteins”. This approach uses a biotin-phenol compound that is turned over by a horse-radish peroxidase (HRP) reporter protein to generate extremely short-lived biotin-phenoxyl radicals and label subcellular proteomes. Labeling specificity arises from the targeting of a HRP fusion protein to subcellular compartments, and we applied this to excitatory synapses (Cijsouw et al, 2018). Our results contributed to the first analyses of the cleft proteome of different synapse types, including synapse-organizing complexes. This progress is now enabling us to determine under this renewal application the synaptic cleft changes that occur in trans-synaptic organizers after psychostimulant exposure.

We will pursue two approaches to attain this goal. First, we will analyze the remodeling of excitatory synapses in striatal slices that occurs after acute cocaine treatment (Javadi-Paydar et al. 2017). This acute effect of cocaine on excitatory synapses agrees with our in vivo finding that a single exposure to cocaine remodels stubby spines in the nucleus accumbens in a manner dependent on the synapse-organizing molecule SynCAM 1 (Giza et al., 2013). We will deliver the proximity labeling reporter HRP-TM (Trans Membrane) that is targeted to postsynaptic sites of excitatory synapses (Cijsouw et al, 2018) into the striatum of mice. We will target medium spiny neurons (MSN), the GABAergic interneurons that make up the vast majority of neurons in this brain region, using the GABA neuron-specific AAV-mDlx (Dimidschstein J et al., 2016) that we already utilize in our group. We will cut slices from the striatum of mice expressing the HRP reporter in MSNs and perform proximity labeling of excitatory inputs to these cells in slices using a permeable biotin-phenol compound. This is followed by affinity purification, on-bead digest, and MS/MS analysis (Cijsouw et al., 2018). We will perform these studies both in wild-type mice and in conditional SynCAM 2 KO mice available in our group. We have selected these KO mice due to genome-wide association studies that linked the gene encoding SynCAM 2, CADM2, to human risk taking and drug abuse (Day et al., 2016; Pasman et al., 2018; Sanchez-Roige et al. 2019). KO mice lacking SynCAM 2 in striatal MSNs will be generated by crossing cKO mice to the Dlx5/6-Cre line (The Jackson Laboratory, line Tg(dlx5a-cre)1Mekk/J). We will analyze how SynCAM 2 loss impacts cocaine-induced remodeling of synapse structure and the molecular makeup of the synaptic cleft, using synaptic labeling (Giza et al, 2013) in conjunction with the proximity labeling approach described above.

As a second approach, we will utilize an in vitro mixed culture system of embryo-derived cortical and MSN neurons that provides for substantially improved MSN differentiation and function compared to MSN monocultures (Penrod et al., 2011). HRP-TM will be expressed in GABAergic neurons, including MSNs that will make up the majority of interneurons in this culture system, using delivery by AAV-mDlx. Neurons will be treated acutely and chronically with increasing amounts of cocaine to establish an in vitro system for psychostimulant-induced synapse remodeling. Molecular changes in synapse organizing molecules of MSNs will be determined using the proximity labeling approach (Cijsouw et al., 2018) and will be correlated with studies of structural changes using immunocytochemistry, as established in our group (Perez de Arce et al., 2015).

Together, the proposed research will provide molecular insights into the synapse-organizing signaling pathways that underlie the changes in neuronal connectivity that are caused by drugs of abuse in the striatum. This can identify permissive and restrictive synapse organizers that serve as novel points of intervention.

Cijsouw T, Ramsey AM, Lam TT, Carbone BE, Blanpied TA, Biederer T (2018) Mapping the proteome of the synaptic cleft through proximity labeling reveals new cleft proteins. Proteomes 6, 48. doi:10.3390/proteomes6040048

Day FR, Helgason H, Chasman DI, Rose LM, Loh PR, Scott RA, Helgason A, Kong A, Masson G, Magnusson OT, Gudbjartsson D, Thorsteinsdottir U, Buring JE, Ridker PM, Sulem P, Stefansson K, Ong KK, Perry JRB (2016) Physical and neurobehavioral determinants of reproductive onset and success. Nat Genet 48:617-623.

Dimidschstein J et al. (2016) A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat Neurosci 19:1743-1749.

Giza JI, Jung Y, Jeffrey RA, Neugebauer NM, Picciotto MR, Biederer T (2013) The synaptic adhesion molecule SynCAM 1 contributes to cocaine effects on synapse structure and psychostimulant behavior. Neuropsychopharmacology 38:628-638.

Javadi-Paydar M, Roscoe RF, Jr., Denton AR, Mactutus CF, Booze RM (2017) HIV-1 and cocaine disrupt dopamine reuptake and medium spiny neurons in female rat striatum. PLoS One 12:e0188404.

Muskiewicz DE, Uhl GR, Hall FS (2018) The role of cell adhesion molecule genes regulating neuroplasticity in addiction. Neural Plast 2018:9803764.

Pasman JA et al. (2018) GWAS of lifetime cannabis use reveals new risk loci, genetic overlap with psychiatric traits, and a causal influence of schizophrenia. Nat Neurosci 21:1161-1170.

Penrod RD, Kourrich S, Kearney E, Thomas MJ, Lanier LM (2011) An embryonic culture system for the investigation of striatal medium spiny neuron dendritic spine development and plasticity. J Neurosci Methods 200:1-13.

Perez de Arce K, Schrod N, Metzbower SW, Allgeyer E, Kong GK, Tang AH, Krupp AJ, Stein V, Liu X, Bewersdorf J, Blanpied TA, Lucic V, Biederer T (2015) Topographic mapping of the synaptic cleft into adhesive nanodomains. Neuron 88:1165-1172.

Sanchez-Roige S, Fontanillas P, Elson SL, Gray JC, de Wit H, MacKillop J, Palmer AA (2019) Genome-Wide Association Studies of Impulsive Personality Traits (BIS-11 and UPPS-P) and Drug Experimentation in up to 22,861 Adult Research Participants Identify Loci in the CACNA1I and CADM2 genes. J Neurosci 39:2562-2572.