Abstract
Marine invertebrate models (squid, sepia, and octopus) made important contributions to description mammals’ nervous system. Being a very simple nervous system relatively easy to be manipulated experimentally and visualized by simple microscope or magnifying glass, the giant synapses at stellate ganglion and the large synaptosomes prepared from the squid photoreceptor neurons served as an attractive model to Histology and Anatomy studies. This sophisticated nervous system has elucidated synaptic transmission in detail with their numerous proteins at presynaptic terminal, synaptic vesicle biogenesis, neurotransmitter secretion, vesicle recycling and, allowed the study of postsynaptic complex with their membranes receptors. However, there are few studies with biochemical and molecular approaches, which lead to a better understanding of their physiological functions and verify operation of such nervous system.
Keywords
- nervous system
- axons and giant synapses
- synaptosomes and hnRNP proteins
1. Introduction
The brain is the control center that transmits the information of neurons to other groups of neurons. A single neuron can be affected simultaneously by excitatory and inhibitory stimuli from one axon or many other axons. Billions of neurons have different functions and constantly check the internal environment and external universe: light, touch, pressure, sound, balance, images, pain, emotion, consciousness. The nervous system from different external stimuli produces a continuous flow of information, memory, learning, and this conscious state throughout life seems infinite.
The nervous system of vertebrate is complex to be studied in individual or small groups of neurons. The cephalopods Coleoidea group (squid, sepia, and octopus), in addition to sea snails and nematodes, which contain few neurons and large structures, are relatively easy to manipulate experimentally and contributed to most of the current knowledge about the nervous system.
In general, these studies can be applicable to nervous system complexes, such as the human system. For example, mammals have a complexity of the Neuropil, an area composed of mostly unmyelinated axons, dendrites, and glial cell processes. For few (μ2) at Neuropil mammals, we find hundreds of bud’s terminals with their corresponding dendritic spines and pre-postsynaptic terminals, where pointing them out individually would be a difficult task.
It was noticed that the Neuropil from squid nervous system also has many pre-postsynaptic terminals such as a mammal. However, these are much larger in the squid nervous system and allow different types of experimental manipulations. In fact, light microscopy cannot resolve synaptic densities and synaptic vesicles, but the active zones in synapses can be observed easily from squid nervous system using low-power electron microscopy.
Thus, the squid nervous system with giant synapses and giant axon system have contributed to the current knowledge of Electrophysiology, Molecular Biology, and Biochemistry and allowed to verify the synaptic transmission and synaptic plasticity.
2. Nature history of Doryteuthis SSP : in the South and North Atlantic
According to the classification by Young [1], cephalopod can be grouped in the subclasses Nautiloidea (nautiluses) and Coleoidea (all the others) or in the general morphology to include in taxonomic descriptions [2].
During the summer in the South and North Atlantic, the mature specimens of
The squid survives chasing food by capturing prey and escaping predators. An ability to accelerate quickly and make sudden changes in the direction of swimming help them to avoid danger. This agility is ensured by the sophisticated nervous system [3, 4] specialized for propellant jets: that pull the water into mantle and then, with the aid of a muscular body wall, which is rapidly contracted, the water is expelled, thus propelling it through the water.
To obtain this muscle contraction result, the squid requires a nervous system that can conduct signals with great speed throughout the body. The optic lobe or “brain” located on each side of the squid head is the control center that transmits the information to the chain of giant nerve cells in the mantle (Figure 1a).
The giant axon can reach 10 cm in length and is about one hundred times than the axon diameter of mammal [5, 6]. The giant axon model made important contributions to the description of axoplasmatic flow mechanisms [7], ion transport across the plasma membrane [8], and neurotransmission [9], and also allowed micro-injections using specific antibodies such a tool for the study of molecules involved in the synaptic function [10, 11]. In addition, the neuronal system contributed to discovery that mRNAs are present in the presynaptic region [12, 13] and new proteins synthesis occurs locally in this region [14].
Here, the stellate ganglion and the large synaptosome from photoreceptor neurons were isolated from squid nervous system and showed a biological and structural organization of brain by biochemical and immunohistochemistry studies.
3. Ultrastructure: giant synapse
Intact stellate ganglion in the mantle was removed (Figure 1b see in pin). The stellate ganglion in seawater can be observed by glass magnification (Figure 1c). In more detail, the presynaptic axon (Pre), postsynaptic axon (Post) and giant nerve fibers (Ax) are seen inFigure 2a.
In light microscopy, cross-sectional region from stellate ganglion shows synaptic contact region between the pre- and postsynaptic terminal at giant synapse when visualized by H&E staining (Figure 2b).
Low-power electron micrograph of same region from stellate ganglion shows synaptic densities and clustering of synaptic vesicles that can be observed in the active zones (Figure 3). The presynaptic (Pre) terminal is lighter than the postsynaptic (Post) terminal and can be characterized by the presence of synaptic vesicles. In the contact areas, the postsynaptic sends digitiform processes and forms the active zones in the limits of interaction between presynaptic terminals. The electron micrograph shows two synaptic densities with clustering of synaptic vesicles in correspondence active zones at the giant synapse (arrows Figure 3).
In general, synapses are local of communication where the neurons pass signals through their axons to postsynaptic target (dendrites, axon or cell body of another neuron, muscle cells, or glandular cells). There are two types of synapses (electrical and chemical) that differ in structure and function. The neurons that communicate through electrical synapses outlets are connected by gap junctions, through which the electrical impulse signals are passed directly from pre- to postsynaptic terminals with high speed. On the other hand, chemical synapses contain the synaptic vesicles at the presynaptic terminal, which carry specific neurotransmitters and have ion channels in the plasma membrane. What differs between chemical and electrical synapses by electrophysiology approach is their impulse speed with a delay feature around 0.5 ms between them, respectively.
Each synaptic vesicles (SV) consists of an apparatus with hundreds of specific proteins to produce fusion of their membranes with the presynaptic membrane and secrete the neurotransmitters at the synapses. It is integrated by corresponding area of neuron, which contains a part of the postsynaptic density (PSD) with ion channel receptors at the postsynaptic membrane for neurotransmitters [10, 15].
The size of synaptic vesicles is variable and dependent of neurotransmitter type [16]. In general, there are two types of vesicles: electron-dense center vesicle and electron-lucent center vesicle. Electron-dense vesicles are subdivided into two types: containing catecholamines (80 nm) and, large synaptic vesicles that contain neuropeptides (200 nm). On the other hand, electron-lucent vesicles have 50 nm of diameter and been carried out with acetylcholine, glycine, GABA, or glutamate. These vesicles are accumulated close at the active zone with 0.5 mm of distance from the plasma membrane.
The membrane proteins (SV) are synthesized in the endoplasmic reticulum granular (REG) and carried through vesicles of the Golgi complex to the presynaptic terminal. The motor proteins (kinesin and dynein) make the transport through microtubules [7] and myosin proteins [17, 18, 19] through actin filaments [20]. The membrane proteins (SV) probably are selected in the primary endosome, which give rise to the synaptic vesicle that to be filled with neurotransmitters.
In summary, when an action potential reaches in the presynaptic terminal least vesicles fuse with specify region at the presynaptic membrane. The action potential in the presynaptic terminal is a very fast process, which have voltage-operated channels allowing rapid and transient entry of calcium (Ca2+) that interacts with several group of vesicular proteins and membrane protein into presynaptic terminal [7, 21, 22]. However, are many proteins involved with endocytosis and exocytosis process of synaptic vesicles.
The calcium triggers fusion of synaptic vesicle with the presynaptic membrane, whereas the central protein involved in fusion of synaptic vesicles is Synaptotagmin protein, which is defined such a calcium sensor due to its C2A domain. The C2A domain through its interaction with calcium undergoes a conformational change, causing it to fuse phospholipids from vesicle and presynaptic membranes. On the other hand, the C2B domain of Synaptotagmin is involved with recycling of synaptic vesicles [23, 24, 25]. Finally, electrophysiology and biochemical approaches of synaptic events have been obtained from the squid nervous system studies, in which specific antibodies and homologous proteins were used for knowledge of synaptic events [10, 11, 15, 26, 27, 28], and also more recently, genetic approach was showed a knockout of the squid pigmentation gene [29]. This method demonstrated efficient gene knockout in the squid
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, 31, 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, 36, 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].
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, 44, 45, 47, 48, 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
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.
6.4 Immunohistochemistry
Fixed, 1-mm transversal slices of optic lobes were included in Paraplast (Oxford Labwase, St. Louis, MO, USA) following the manufacturer’s instructions. Microtome slices of 10 μm were cut, transferred to glass slides, de-parafinized, and rehydrated by standard procedures. Slices were incubated in PBS pH 7.4 containing 0.1 M glycine for 30 min at 4°C to block aldehyde groups and then washed three times for 10 min in PBS. They were then incubated in the dark at room temperature for 30 min in methanol containing 0.9% hydrogen peroxide solution to inhibit endogenous peroxidase activity, followed by washing in PBS. The samples were permeabilized and blocked by incubation in PBS containing 1% Triton X-100, 3% BSA, and 0.5% sheep serum and then incubated for 1 h with primary antibodies in PBS containing 0.1% Triton X-100, 3% BSA, and 0.5% sheep serum. After washing in PBS containing 0.1% Triton X-100, the slices were incubated for 1 h with secondary antibodies conjugated to horse radish peroxidase (KPL, Gaithersburg, MD, USA) diluted 1:400 in PBS containing 0.1% Triton X-100, 3% BSA, and 0.5% sheep serum and developed using 3,3-diaminobenzidina (DAB) as substrate.
6.5 Immunofluorescence
Synaptosomes in suspension were fixed in 1% paraformaldehyde for 6 hr. at room temperature, adhered to glass microscope slides by incubation for 1 hr. After gentle washing with PBS, slides were incubated for 30 min in 0.1 M glycine, 0.3% Triton X-100 in PBS, pH 7.4, and washed and blocked in 1 mg/ml BSA, 1% goat serum, and 1% Triton X-100 for 1 hr. at room temperature. The slides were washed with PBS containing 0.3% Triton X-100 and incubated with primary antibody in PBS containing 1 mg/ml BSA, 1% goat serum, and 0.3% Triton X-100 for 2 hrs. at room temperature. The slides were washed in PBS and incubated with appropriate secondary antibody for 1 hr. and washed again. The slides were mounted in Fluoromount G (EMS) diluted 2:1 in PBS and examined on a Zeiss 510 confocal microscope.
Acknowledgments
Dr. Gabriel Sarti Lopes and Dr. Jorge E. Moreira of University of São Paulo, FMRP/USP and Dra. Janaina Brusco from University of British Columbia, Can. Financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) are acknowledged. Special thanks to Dr. Roy E. Larson of University of São Paulo, FMRP/USP (past advisor). Infrastructure support from Marine Biology Center at University of São Paulo (CEBIMar-USP) and Marine Biological Laboratory, Woods Hole-US. Special thanks to Patricia Horta, PhD, IFSP, that revised this chapter.
Conflict of interest
The author declares that there is no conflict of interests regarding the publication of this chapter.
References
- 1.
Young JZ. Structure of nerve fibres in sepia. Journal of Physiology Land. 1935; 83 :27P-28P - 2.
Roper CF, Voss GL. Guidelines for taxonomic descriptions of cephalopod species. Memoirs. National Museum of Victoria. 1983; 44 :49-63 - 3.
Bullock TH. Comparative neuroethology of startle, rapid escape, and giant fibre-mediated responses. In: Eaton RC, editor. Neural Mechanisms of Start I Behavior. New York and London: Plenum Press; 1984. pp. 1-13 - 4.
Mackie GO. Giant axons and the control of jetting in the squid Loligo and the jellyfishAglantha . Canadian Journal of Zoology. 1990;68 :799-805 - 5.
Gilly WF, Hopkins B, Mackie GO. Development of giant motor axons and neural control of escape responses in squid embryos and hatchlings. Biology I Bulletin. 1991; 180 :209-220 - 6.
Roper CFE, Ross KJ. The giant squid. Science. 1982; 246 :96-105 - 7.
Vale RD, Reese TS, Sheetz MP. Identification of a novel force-generating protein kinesin involved in microtubule-based motility. Cell. 1985; 42 :39-50 - 8.
Katz B, Miledi R. Membrane noise produced by acetylcholine. Nature. 1970; 226 :962-963 - 9.
Llinás R, Sugimori M, Simon SM. Transmission by presynaptic spike-like depolarization in the squid giant synapse. Proceedings of the National Academy of Sciences of the United States of America. 1982; 79 :2415-2419 - 10.
Fukuda M, Moreira JE, Lewis FMT, Sugimori M, Niinobe M, Mikoshiba K, et al. Role of the C2b domain of synaptogamin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal. Proceedings of the National Academy of Sciences USA. 1995; 92 :10703-10707 - 11.
Sugimori M, Tong C, Fukuda M, Moreira JE, Kojima T, Mikoshiba K, et al. Presynaptic injection of syntaxin-specific antibodies block transmission in the squid giant synapse. Neuroscience. 1998; 86 :39-51 - 12.
Gioio AE, Lavina ZS, Jurkovicova D, Zhang H, Eyman M, Giuditta A, et al. Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA. The European Journal of Neuroscience. 2004; 20 :865-872 - 13.
Giuditta A, Kaplan BB, van Minnen J, Alvarez J, Koenig E. Axonal and presynaptic protein synthesis: New insights into the biology of the neuron. Trends in Neurosciences. 2002; 25 :400-404 - 14.
Martin KC, Casadio A, Zhu H, Yaping E, Rose JC, Chen M, et al. Synapse-specific, long-term facilitation of aplysia sensory to motor synapses: A function for local protein synthesis in memory storage. Cell. 1997; 91 :927-938 - 15.
Mikoshiba K, Fukuda M, Moreira JE, Lewis FMT, Sugimori M, Niinobe M, et al. Role of the C2b of synaptogmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal. Proceedings of the National Academy of Sciences USA. 1995; 92 :10708-10712 - 16.
Burns ME, Augustine GJ. Synaptic structure and function: Dynamic organization yields architectural precision. Cell. 1995; 83 :187-194 - 17.
Calliari A, Sotelo-Silveira J, Costa MC, Nogueira J, Cameron LC, Kun A, et al. Myosin Va is locally synthesized following nerve injury. Cell Motility and the Cytoskeleton. 2002; 51 :169-176 - 18.
Ohashi S, Koike K, Omori A, Ichinose S, Ohara S, Kobayashi S, et al. Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor. The Journal of Biological Chemistry. 2002; 277 :37804-37810 - 19.
Sotelo-Silveira JR, Calliari A, Cardenas M, Koenig E, Sotelo JR. Myosin Va and kinesin II motor proteins are concentrated in ribosomal domains (periaxoplasmic ribosomal plaques) of myelinated axons. Journal of Neurobiology. 2004; 60 :187-196 - 20.
Bearer EL, Debirgis JA, Boduer RA, Aw K, Reese TS. Evidence for miosin motors on organelles in squid axoplasm. Proceedings of the National Academy of Sciences USA. 1993; 90 :11252-11256 - 21.
Edwards RH. The transport of neurotransmitters into synaptic vesicles. Current Opinion in Neurobiology. 1992; 2 :586-594 - 22.
Régnier-Vigouroux A, Tooze AS, Huttner WB. Newly synthesized synaptophysin is transported to synaptic-like microvesicles via constitutive secretory vesicles and the plasma membrane. The EMBO Journal. 1991; 10 :3589-3601 - 23.
Betz WJ, Bewick GS. Optical monitoring of neurotransmitter release and synaptic vesicle recycling at the frog neuromuscular junction. Journal of Physiology (London). 1993; 460 :287-309 - 24.
Hata Y, Slaughter CA, Sudhof TC. Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature. 1993; 366 :347-351 - 25.
Ryan TA, Smith SJ. Vesicle pool mobilization during action potential firing at hippocampal synapses. Neuron. 1995; 14 :983-989 - 26.
Gainer H, Galdlant PE, Gould R, Pant HC. Biochemistry and metabolism of the squid Giant axon. Current Topics in Membranes and Transport. 1984; 22 :57-90 - 27.
Llinás RR, Sugimori M, Lang EJ, Morita M, Fukuda M, Niinobe M, et al. The inositol high-polyphosphate series block synaptic transmission by preventing vesicular fusion: A squid giant synapse. Proceedings of the National Academy of Sciences USA. 1994; 91 :12990-12993 - 28.
Marsal J, Ruiz-Montacell B, Blasi J, Moreira JE, Contreras D, Sugimori M, et al. Block of transmitter release by botulinium C1 action on syntaxin at the squid giant synapse. Proceedings of the National Academy of Sciences USA. 1997; 94 :14871-14876 - 29.
Karen C, Diaz JF, Quiroz KM, Koenig NA, Albertin CB, Rosenthal JJC. Highly efficient knockout of a squid pigmentation gene. Current Biology. 2020; 30 :3484-3490 - 30.
Lico DT, Lopes GS, Brusco J, Rosa JC, Gould RM, De Giorgis JA, et al. A novel SDS-stable dimer of a heterogeneous nuclear ribonucleoprotein at presynaptic terminals of squid neurons. Neuroscience. 2015; 300 :381-392 - 31.
Lico DTP, Rosa JC, DeGiorgis JA, deVasconcelos EJR, Casaletti L, Tauhata SBF, et al. A novel 65 kDa RNA-binding protein in squid presynaptic terminals. Neuroscience. 2010; 166 :73-83 - 32.
Lopes GS, Brusco J, Rosa JC, Larson RE, DTP L. Selectively RNA interaction by a hnRNPA/B-like protein at presynaptic terminal of squid neuron. Invertebrate Neuroscience. 2020; 20 :14 - 33.
Lopes GS, Lico DTP, Rocha RS, Oliveira RR, Sebollela AS, Paco-Larson ML, et al. A phylogenetically conserved hnRNP type A/B protein from squid brain. Neuroscience Letters. 2019; 696 :219-224 - 34.
Haghighat N, Cohen RS, Pappas GD. Fine structure of squid ( Loligo pealei ) optic lobe synapses. Neuroscience. 1984;13 :527-546 - 35.
Budelmann BU. Cephalopod sense organs, nerves and the brain: Adaptations for high performance and life style. Marine and Freshwater Behaviour and Physiology. 1994; 25 :13-33 - 36.
Young JZ. The Anatomy of the Nervous System of Octopus vulgaris. Oxford: Clarendon Press; 1971 - 37.
Young JZ. The central nervous system of Loligo. I. the optic lobe. Philosophical Transactions of Royal Society of London B. 1974; 267 :263-302 - 38.
Gabriel LS, Janaina B, Jose RC, Roy LE, Diego LTP. Selectively RNA interaction by a hnRNPA/B-like protein at presynaptic terminal of squid neuron. Invertebrate Neuroscience. 2020; 20 :1-14 - 39.
Dimasi P et al. Modulation of p-eIF2α cellular levels and stress granule assembly/disassembly by trehalose. Scientific Reports. 2017; 7 :44088 - 40.
Mahboubi H, Stochaj U. Cytoplasmic stress granules: Dynamic modulators of cell signaling and disease. Biochimica et Biophysica Acta - Molecular Basis of Disease. 2017; 1863 :884-895 - 41.
Aulas A, Vande Velde C. Alterations in stress granule dynamics driven by TDP-43 and FUS: A link to pathological inclusions in ALS? Frontiers in Cellular Neuroscience. 2015; 9 :423 - 42.
Heraviab YB, Van Broeckhovenab C, der Zee J. Stress granule mediated protein aggregation and underlying gene defects in the FTD-ALS spectrum. Neurobiology of Disease. 2020; 134 :104639 - 43.
Polymenidou M, Cleveland DW. Biological spectrum of amyotrophic lateral sclerosis prions. Cold Spring Harbor Perspectives in Medicine. 2017; 7 :a024133 - 44.
Ramaswami M, Taylor JP, Parker R. Altered ribostasis: RNA-protein granules in neurodegenerative disorders. Cell. 2013; 154 :727-736 - 45.
Zhang K, Daigle JG, Cunningham KM, Coyne AN, Ruan K, Grima JC, et al. Stress granule assembly disrupts nucleocytoplasmic transport. Cell. 2018; 173 :958-971.e917 - 46.
Li Y et al. RBM45 homo-oligomerization mediates association with ALS-linked proteins and stress granules. Scientific Reports. 2015; 5 :14262 - 47.
Ash PEA et al. Pathological stress granules in Alzheimer’s disease. Brain Research. 2014; 1584 :52-58 - 48.
Kim HJ, Kim NC, Wang Y-D, Scarborough EA, Moore J, et al. Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature. 2013; 495 :467-473 - 49.
Wolozin B. Regulated protein aggregation: Stress granules and neurodegeneration. Molecular Neurodegeneration. 2012; 7 (1):56 - 50.
Gabriel S. Lopes and Lico DT P. Biochemical and subcellular characterization of a squid hnRNPA/B-like protein in osmotic stress activated cells reflects molecular properties conserved in this protein family. Molecular Biology Reports. 2022. DOI: 10.1007/s11033-022-07260-0 - 51.
Pekkurnaz G, Fera A, Zimmerberg-Helms J, DeGiorgis JA, Bezrukov L, Blank PS, et al. Isolation and ultrastructural characterization of squid synaptic vesicles. Biology Bulletin. 2011; 220 :89-96