FXYD6 at VGLUT1 synapses

Complementing the previous findings on the localization of FXYD6 in neuronal cell bodies and dendrites, my proteomic analysis of VGLUT1^VENUS synaptosomes isolated by

FASS indicated that FXYD6 is present at VGLUT1 synapses. Analysis by Western blotting confirmed the enrichment of FXYD6 in the sorted VGLUT1^VENUS

Figure 27

synaptosomes, indicating that even quantifications based on relatively low spectral counts may correctly reflect the relative distribution of a protein between two samples ( A). Consistent with the enrichment in VGLUT1^VENUS synaptosomes and with its potential effect on the Na⁺/K⁺

Figure 27

-ATPase at the neuronal plasma membrane FXYD6 was found to be enriched in synaptic plasma membrane fractions (LP1B) upon subcellular fractionation of forebrain tissue (

B). Furthermore, Western blot analyses of brain region specific tissue homogenates revealed that FXYD6 is expressed throughout the brain. This result is consistent with our observations in immunostainings of brain sections as well as the Western blot analyses performed in earlier studies (Kadowaki et al., 2004).

Using immunofluorescence staining of cultured neurons and brain sections, we confirmed earlier observations that FXYD6 is present on the plasma membrane of neuronal cell bodies and dendrites (Figure 28 and Figure 29). FXYD6 does not seem to be expressed in GFAP positive astrocytes cocultured with the neurons from the hippocampus (Figure 28B).

Using Immuno-EM we localized FXYD6 at the plasma membrane of axons, axon terminals, and to a lesser extend, also of synaptic vesicles (Figure 29). Importantly, we found FXYD6 to be colocalized with presynaptic VGLUT1 in cultured hippocampal neurons and in the hippocampus and cortex in situ. Father analyses revealed that FXYD6 is also present at VGLUT2 expressing synapses in cultured hippocampal neurons and that in the hippocampus it is not present at axonal varicosities of inhibitory neurons (Figure 29). Together these results provide evidence for the presence of FXYD6 at VGLUT1synapses, which I originally inferred from our analysis of FASS purified VGLUT1^VENUS

My results indicate that FXYD6 is localized to VGLUT1 synapses and is expressed preferentially in excitatory neurons. This substantiates the usefulness of my FASS protocol for the identification of novel components of VGLUT1 synapses.

synaptosomes. Additionally we could observe that while FXYD6 is strongly expressed in the VGLUT1 expressing granule cells of the cerebellum, the inhibitory Purkinje cells do not express FXYD6 (data not shown).

These results, together with the segregation of GAD65/67 and FXYD6 stainings in the hippocampus, indicate a preferential localization of FXYD6 in excitatory neurons.

Furthermore, the fact that FXYD6 was localized in presynaptic terminals and dendrites of neurons raises the possibility that FXYD6 functions in regulating the transport properties of Na⁺/K⁺

Given that FXYD6 has a differential effect on the properties of different Na -ATPases in subcellular compartments involved in excitatory neurotransmission. Future studies will have to address the potential role of FXYD6 on pre-synaptic and post-pre-synaptic aspects of pre-synaptic transmission.

+/K⁺ -ATPase isozymes it will be interesting to correlate their respective cellular and subcellular

localization. Furthermore, phosphorylation of FXYD isoforms can alter their effect on the Na⁺/K⁺-ATPase (Arystarkhova et al., 2002). In synaptic transmission, local signaling cascades might therefore allow for regional adaptations of the transport properties of the Na⁺/K⁺-ATPase through modification of FXYD proteins. Finally it is known that Na⁺/K⁺ -ATPase isozymes also display differential expression patterns within the brain(Moseley et al., 2003; Richards et al., 2007). Together, the differential distribution of FXYD proteins and Na⁺/K⁺

4.6 TPD52

-ATPase isozymes may allow for complex fine-tuning of the ion-homeostasis of neurons.

The tumor protein D52 (also named CRHSP-28,CSPP28 or R10) was initially identified because of its overexpression in several human cancers and cancer cell lines (Byrne et al., 1996). Since then multiple lines of evidence implicate TPD52 in secretory processes:

(i) TPD52 functions in Ca²⁺-regulated exocytosis in secretory cells in the digestive system, namely the gastric chief cells and pancreatic acinar cells (Groblewski et al., 1996;

Parente et al., 1996; Thomas et al., 2001). (ii) TPD52 is highly expressed in mature B-cells and plasma cells where it has been implicated in the regulation secretory processes involving the Ca²⁺

TPD52 mRNA and protein are found in the brain and immunofluorescence data indicate that TPD52 is present in as yet unidentified granule rich cells in the hippocampus (Chew et al., 2008; Groblewski et al., 1999; Parente et al., 1996). Additional evidence for the expression of TPD52 was presented by functional correlation of microarray based gene expression data, which identified TPD52 as a gene, whose expression is highly correlated with that of genes that encode neuronal and synaptic proteins (Oldham et al., 2008). In my proteomic analysis of FASS samples, TPD52 was enriched 3.33-fold in sorted VGLUT1

-dependent binding of TPD52 to AnnexinVI (Tiacci et al., 2005). (iii) A recent study implicated TPD52 in the regulation of the trafficking of lysosomal proteins to the plasma membrane (Thomas et al., 2010). (iv) TPD53 (D53) a close homolog of TPD52 can bind to neuronal SNARE proteins in vitro and can be immunoprecipitated with synaptobrevin-2 from cell extracts (Proux-Gillardeaux et al., 2003) (v) All members of the TPD52 protein family can interact with MAL2 in yeast-two hybrid interaction assays, and MAL2 has recently been identified as a specific component of VGLUT1 containing synaptic vesicles (Grønborg et al., 2010).

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synaptosomes as assessed by spectral counting. Taking into account all the evidence mentioned above, it is a plausible hypothesis that TPD52 can localize to and function at VGLUT1 synapses.