**4. Immunohistochemistry and immunofluorescence studies: squid optic lobe and synaptosome**

The complexity of subcellular domains in neurons and their distances of nucleus demand a spatial and temporal control of protein synthesis. We have identified a novel member of hnRNP A/B type, of 65 kDa, in the presynaptic terminal of squid neuron [30–32]. Squid p65 was phylogenetically conserved hnRNP type A/B protein in mammals [33]. In this scenario, these hnRNPs have emerged as major components of mechanisms for the local protein synthesis and synaptic plasticity.

To assess their presynaptic terminal location in squid photoreceptors neurons, the immunohistochemistry of slices from optic lobe was incubated with the anti-synaptic vesicle glycoprotein 2A (ɑ-SV2) antibody and developed by peroxidase-DAB (see in method). The ɑ-SV2 antibody recognizes the corresponding bands in the outer plexiform layer (**Figure 4a**), which is a synaptic connection region [34]. This image was similar to those previously obtained with the Synaptotagmin antibody [31]. These data showed an anatomy is highly complex with outer cortical layers and a central medulla from squid optic lobe [35–37].

To further indicate the subcellular localization of hnRNPs is involved in presynaptic terminal localization in squid photoreceptors neurons, the synaptosomes isolated from optic lobes were double probed with ɑ-SV2 antibody and anti-squid ribonucleoprotein motif 2 (ɑ-sqRNP2) antibody raised in rabbits [30]. Immunofluorescence microscopy of synaptosomes showed intense granular staining with both antibodies frequently close to the plasma membrane, suggesting a spatial relationship between both proteins to clustering synaptic vesicle at the presynaptic terminal (**Figure 4b**).

In fact, hnRNP protein shuttles mature mRNA from nucleus to the cytoplasm and are also involved in packaging mRNAs into cytoplasmic granule transport, which have been more clearly evidenced in dendrite cells. However, the exact function of hnRNPs in the presynaptic terminal has yet to be clarified [31, 38].

### **5. hnRNP proteins and degenerative diseases**

Several cellular compartments not enclosed by membranes are called ribonucleoprotein granules, due to the high concentration of proteins and mRNAs (mRNPs). For example, mRNPs can be found in the nucleus in Cajal bodies, paraspeckles, speckles, etc., but they can also be found in the cytoplasm, such as stress granules and processing bodies (Pbodies).

Stress granules are cytoplasmic complexes made up of proteins and RNA, found in most cell types in culture (from yeast to humans), and are formed under specific conditions of cellular stress [39]. *In vitro* experiments with different conditions can induce the formation of stress granules such as lack of nutrients, heating, protein complex and protein degradation inhibitors (proteasome), genotoxic drugs (such as UV radiation), and drugs that cause oxidative stress (sodium arsenite) or osmotic

*The Biological and Structural Organization of the Squid Brain DOI: http://dx.doi.org/10.5772/intechopen.107217*

#### **Figure 4.**

*Immunolocalization of presynaptic terminal in the optic lobe "chickpeas-like." a) Immunohistochemistry of the 10 nm slice through the squid optic lobe labeled with anti-vesicle protein 2 antibody (ɑ-SV2) and secondary antibody by the peroxidase-DAB (method described by [31]). The arrows indicate the immunopositive bands for both antibodies in the outer plexiform layer (opx). The morphological layers of the optic lobe cortex are indicated to the right: Outer nuclear (on), outer plexiform (opx), inner nuclear (in), inner plexiform (ipx), and mononuclear (mn) layers with optic nerve terminals at the outer plexiform layer (opx) from optic lobe cortex. Scale bars 100 μm. b) Confocal images of two representative synaptosomes that were double immunolabeled with anti-sqRNP2 antibody (ɑ-sqRNP2, green) and anti-vesicle protein 2 antibody (ɑ-SV2, red). The merged images show yellow where overlap occurs. Scale bars 2 μm.*

agent (sorbitol) [39, 40]. Many studies showed an association of mRNPs to neurodegenerative diseases [41–45].

Of course, stress granules are generated as a cellular response to a physiologically unfavorable environment, and their prevalence in neurological disorders suggests that their formation in affected neurons is, at least initially, a cytoprotective response to disease-associated stress. However, the persistence of stress granules can contribute to the development of several degenerative diseases, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and some myopathies and neurodegenerative diseases [46].

The mutations in RNA-binding proteins appear to increase the propensity of these proteins to aggregate and form stress granules. For example, diseases linked to mutations promote aggregation in the proteins such as TDP-43 (TAR DNA-binding protein 43) and FUS (Fused in Sarcoma) [43–45, 47–49].

In previous studies [30, 50] showed molecular and biochemical evidence that p65 is a dimer resistant to SDS treatment, composed of two protein subunits of the hnRNP A/B type with molecular weight around 37 kDa, and this propensity of these proteins to aggregate and form stress granules involves endogenous modification factors. Nevertheless, it did not clear show how such endogenous factors could act to produce alterations in hnRNP A/B-like protein that induces dimerization. Immunohistochemical studies demonstrate the presence of p65 in presynaptic nerve terminals and its propensity to oligomerize led us to further investigate their cellular and molecular properties in presynaptic nerve terminals [50].

In conclusion, based on these points presented here, we suggest that hnRNP A/B protein could be a link between local RNA processing and synaptic function at the presynaptic terminal. Understanding this can bring insights into evolution of several neurodegenerative diseases and verify if they resemble the function performed in vertebrates.

This brief summary has shown that the cephalopods have structure's power that served in comparative studies and useful alternative to invertebrate experimental research that, in general, is applicable to mammalian systems. However, are few studies with biochemical and molecular approach, which could lead to a better understanding of their physiological functions.

### **6. Materials and methods**

#### **6.1 Animals and tissue preparation**

The optic lobes were dissected from freshly killed *D. pealei* obtained from the Marine Biological Laboratory in Woods Hole or from *D. plei* obtained from the Centro de Biologia Marinha-CEBIMar, University of São Paulo, São Sebastião, Brazil. For immunohistochemistry and immunofluorescence procedures freshly dissected optic lobes are from *D. ssp* [31]. The synaptosomes (isolated nerve terminals from photoreceptor cells) were prepared from tissue according to Pekkurnaz et al. [51] with slight modifications. Briefly, optic lobes were quickly dissected from squid onto ice-cold Petri dishes and weighed. Each g of tissue was homogenized in 5 ml of ice-cold homogenization buffer (HB) (1.0 M sucrose in 20 mM Tris-HCl, pH 7.4) in a Wheaton glass homogenizer with a loose-fitting pestle, by 10–15 gentle strokes. The homogenate was spun at 1000xg for 11 min at 4 °C and then spun at 13,000xg for 45 min. The floating synaptosome layer was carefully decanted into a small Petri dish, gently washed in HB and resuspended in 0.5 ml of HB.

#### **6.2 Antibodies**

Anti-squid ribonucleoprotein motif 2 (ɑ-sqRNP2) antibody was raised in rabbits. The ɑ-sqRNP2 antibody raised in rabbits against the synthetic peptide CLFIGGLSYDTNEDTIK corresponds to an internal sequence determined by mass spectrometry from tissue-purified p65 [30]. The rabbit serum after inoculation was purified on a HiTrap Recombinant Protein A column (GE Health Science, Chalfont St.Giles, UK) by Fast protein liquid chromatography (ÄKTAFPLC system). The monoclonal anti-synaptic vesicle glycoprotein 2A (ɑ-SV2) antibody (64,051, Invitrogen, Carlsbad, CA) was raised in mouse and secondary antibodies conjugated to Alexa 488 for immunofluorescence from Molecular Probes (Invitrogen, Carlsbad, CA).

## **6.3 Ultrastructure**

The stellate ganglion was removed from squid mantle on the seawater, fixed by immersion in 2% formaldehyde plus 2% glutaraldehyde in buffered calcium-free seawater, postfixed in osmium tetroxide, stained with uranium acetate, dehydrated, and embedded in Araldite plastic CY212 (EMS). Ultra-thin sections in carbon-coated single-slot grids were contrasted with uranyl acetate and lead citrate. Electron micrographs were taken at an initial magnification of x14,000 and photographically enlarged to a magnification of x35,000.
