Cathepsin L coexists with Cytotoxic T lymphocyte Antigen 2 alpha in distinct regions of the mouse brain

G Model ACTHIS-51114; No of Pages ARTICLE IN PRESS Acta Histochemica xxx (2016) xxx–xxx Contents lists available at ScienceDirect Acta Histochemica journal homepage: www.elsevier.de/acthis Cathepsin L coexists with Cytotoxic T-lymphocyte Antigen-2 alpha in distinct regions of the mouse brain Claudius Luziga a,∗ , Bui Thi To Nga b , Gabriel Mbassa a , Yoshimi Yamamoto c a b c Department of Veterinary Anatomy, Sokoine University of Agriculture, Morogoro, Tanzania Department of Veterinary Pathology, Vietnam National University of Agriculture, Viet Nam Laboratory of Biochemistry and Radiation Biology, Department of Veterinary Sciences, Yamaguchi University, Yamaguchi 753-8515, Japan a r t i c l e i n f o Article history: Received 23 May 2016 Received in revised form 12 August 2016 Accepted 17 August 2016 Available online xxx Keywords: Cathepsin L CTLA-2␣ Immunofluorescence Brain Mouse a b s t r a c t Cathepsins B and L are two prominent members of cystein proteases with broad substrate specificity and are known to be involved in the process of intra- and extra-cellular protein degradation and turnover The propeptide region of cathepsin L is identical to Cytotoxic T-lymphocyte antigen-2␣ (CTLA-2␣) discovered in mouse activated T-cells and mast cells CTLA-2␣ exhibits selective inhibitory activities against papain and cathepsin L We previously demonstrated the distribution pattern of the CTLA-2␣ protein in mouse brain by immunohistochemistry, describing that it is preferentially localized within nerve fibre bundles than neuronal cell bodies In the present study we report colocalization of cathepsin L and CTLA-2␣ by double labeling immunofluorescence analysis in the mouse brain In the telencephalon, immunoreactivity was identified in cerebral cortex and subcortical structures, hippocampus and amygdala Within the diencephalon intense colocalization was detected in stria medullaris of thalamus, mammillothalamic tract, medial habenular nucleus and choroid plexus Colocalization signals in the mesencephalon were strong in the hypothalamus within supramammillary nucleus and lateroanterior hypothalamic nucleus while in the cerebellum was in the deep white matter, granule cell layer and Purkinje neurons but moderately in stellate, and basket cells of cerebellar cortex The distribution pattern indicates that the fine equilibrium between synthesis and secretion of cathespin L and CTLA-2␣ is part of the brain processes to maintain normal growth and development The functional implication of cathespin L coexistence with CTLA-2␣ in relation to learning, memory and disease mechanisms is discussed © 2016 Published by Elsevier GmbH Introduction Several kinds of proteolytic enzymes of mammalian proteases have been identified including aspartic, cysteine, metallo, serine and threonine (Rawlings et al., 2014) Cathepsins are cysteine proteases belonging to the papain subfamily They are predominantly endopeptidases located intracellularly in endolysosomal vesicles Various types of cathepsins have been discovered including cathepsin B, D, H, L, S and P (Barrett et al., 1981; Maubach et al., 1997) Cathepsins B, L, and H are found in most cell types and body tissues where they regulate diverse normal biological processes such as cell death, proliferation, migration, invasion and protein turnover (Barrett et al., 1981; Reddy et al., 1995; Maubach et al., 1997; Deussing et al., 2002; Cowan et al., 2005) Cathepsin L in secretory vesicle has been demonstrated for production of active ∗ Corresponding author E-mail address: luziga@suanet.ac.tz (C Luziga) peptides required for cell to cell communication in the nervous and endocrine systems (Funkelstein et al., 2010) The expression of some cathepsins is high and regulated in specific cell types Cathepsin B and L are expressed constitutively and thought to participate in protein turnover and diseases Studies in mice deficient in cathepsin B or L have indicated a role for the cathepsins in normal brain development Mice deficient in both cathepsin L and B show brain atrophy due to massive apoptosis of cerebral and cerebellar neurons (Felbor et al., 2002) However, prolonged activation of cathepsin B is associated with neuronal degeneration in Alzheimer’s disease (Callahan et al., 1998; Nixon, 2000) while inhibition results in reduction of brain ␤-amyloid peptides and significant improvement in memory in a mouse model of Alzheimer’s disease (Hook et al., 2009) Similarly, gene-expression profiling by cDNA microarrays also show that CTLA-2␣ is highly expressed in mice brain tissues susceptible to cerebral malaria (Delahaye et al., 2006) Regulation of cathepsin activity appears to have a significant role in health and disease http://dx.doi.org/10.1016/j.acthis.2016.08.003 0065-1281/© 2016 Published by Elsevier GmbH Please cite this article in press as: Luziga, C., et al., Cathepsin L coexists with Cytotoxic T-lymphocyte Antigen-2 alpha in distinct regions of the mouse brain Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.08.003 G Model ACTHIS-51114; No of Pages ARTICLE IN PRESS C Luziga et al / Acta Histochemica xxx (2016) xxx–xxx Structural information indicates that cathepsins consist of a signal peptide, a propeptide, and a catalytic domain which is a mature proteolytically active enzyme (Mach et al., 1994; Ménard et al., 1998; Turk et al., 2012) The propeptides of some proteases are reported to be potent inhibitors of the proteases from which they were derived The structure of Cytotoxic T-lymphocyte antigen2 alpha (CTLA-2␣) is homologous to the proregion of cathepsin L (Denizot et al., 1989; Yamamoto et al., 2002) and that CTLA-2␣ is a potent inhibitor of cathepsin L − like cystine proteases (Delaria et al., 1994; Carmona et al., 1996; Deshapriya et al., 2010; Kurata et al., 2003) Other propeptide-like cysteine proteinase inhibitor proteins homologous to CTLA-2␣ have been identified in other organisms including the Bombyx cysteine protease inhibitor (BCPI) identified in Bombyx mori (Yamamoto et al., 1999a, 1999b; Kurata et al., 2001) and the crammer peptide (CG10460 gene product) found in Drosophila melanogaster (Comas et al., 2004) The Drosophila crammer gene (CG10460) which is homologous to mouse CTLA-2␣ gene, was found to be expressed in Drosophila glial cells and mushroom bodies, the Drosophila olfactory memory centre, that form a prominent bilateral structure of the insect brain The concentration of expressed crammer is shown to be critical for the establishment of long-term memory, suggesting a role of this inhibitor in memory formation through regulation of cathepsin activity (Comas et al., 2004) In the hippocampus concurrent inhibition of multiple cysteine proteases induces a decrease in long-term formation but not short-term spatial memory in mouse (Dash et al., 2000) In this context, information on the colocalization of cathepsin L (a family of cysteine proteinases) with CTLA-2␣ in the central nervous system is pertinent to several aspects of learning, memory establishment and diseases This study was therefore aimed at examining simultaneous localization of cathepsin L and CTLA2␣ in the mouse brain by double labeling immunofluorescence microscopy tion and preparation of antiserum were performed as previously described (Takahashi et al., 1993) The polyclonal anti-cathepsin L antibody against rabbit cathepsin L protein and anti-CTLA-2␣ antibody against chicken CTLA-2␣ protein were obtained through affinity chromatography column with recombinant cathepsin L and CTLA-2␣ conjugated resins respectively The specificity of the purified antibodies was characterized as previously described (Bui et al., 2015) Materials and methods 3.1 Colocalization pattern of cathepsin L with CTLA-2˛ protein in various structures of the mouse brain 2.3 Double immunofluolencence microscopy Sections were deparaffinized and hydrated in a consecutive series of xylene and ethanol to phosphate-buffered saline (0.01 M PBS-pH 7.4) Endogenous peroxidase activity was blocked by immersing the tissue sections in a solution of 0.3% v/v hydrogen peroxide in distilled water for 30 at room temperature (RT) and then washed (3 × min) in PBS Afterwards, the sections were blocked with 10% goat normal serum for 30 at RT to avoid nonspecific labeling The sections were incubated with a mixture of primary antibodies containing both cathepsin L and CTLA-2␣ (1:500) IgG and IgY in PBS, pH 7.4 for 24 h in a dark, humid chamber at ◦ C For negative control, 10% goat normal serum was applied to some sections in place of primary antibody Sections were then washed (3 × min) in PBS followed by incubation with a mixture of Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (FITC) and Alexa Fluor® 594-conjugated goat anti-chicken IgY (TRITC) at a dilution of 1:100 (Molecular Probes, Inc Eugene, USA) for h at RT At the end of incubation, the sections were washed (3 × min) in PBS and mounted Immunoreactivity was examined using the BZ9000E HS all-in-one Fluorescence Microscope (KEYENCE, Japan) Morphological structures refer to the neuron-anatomical atlas from Paxinos and Franklin (2001) (Table 1) Results 2.1 Animals and tissue preparation A total of 10 mice were kept in a room at 19–21 ◦ C temperature, with free access to food and water All experimental procedures were performed according to the guide for protection and control of animal experimentation in Japan Permission to use animals in experiments was approved by the Animal Protection and Control committee of Yamaguchi University Ten adult male and female mice five in each, aged 12 months were studied, sagittal and coronal cutting planes were prepared The mice were anesthetized with sodium pentobarbital (70 mg/kg) by intraperitoneal injection and transcardiac perfusion with 0.01 M phosphate buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde (PFA; Sigma-Aldrich, St Louis, MO) in 0.1 phosphate buffer (PB; pH 7.4) Brain tissues were dissected and postfixed in 4% PFA for h at ◦ C The tissues were then processed through graded ethanol series to paraffin wax and sectioned at a thickness of ␮m using a microtome, then used for immunofluorescence analysis 2.2 Generation of antibodies Recombinant cathepsin L and CTLA-2␣ were purified using methods described previously with minor modifications Affinitypurified rabbit anti-cathepsin L IgG and chicken anti-CTLA-2␣ IgY were generated In brief, antiserum against cathepsin L was obtained by immunizing rabbit against recombinant cathepsin L Antiserum against CTLA-2␣ was obtained by immunizing chicken against recombinant CTLA-2␣ (Camenisch et al., 1999) Immuniza- Immunofluorescence evaluation of cathespin L colocalization with CTLA-2␣ was performed on sagittal and coronal sections of the mouse brain Cathepsin L with CTLA-2␣ displayed a region-specific colocalization, being strongly present in some brain structures but not detectable in others Strong labeling was observed in the external capsule (ec); corpus callosum (cc); fimbria of hippocampus (fi); interneurons in Cornu Ammonis 2, fields of hippocampus; stria medullaris (sm); fibres of mammillothalamic tract (mt) and anterior commissure (ac) Moderate labeling was detected in neocortex; intermediate part of lateral septal nucleus (LSI) and in majority of thalamic nuclei including anterodorsal thalamic nucleus (AD); central part of mediodorsal thalamic nucleus (MDC) and medial preoptic area (MPA) in sagittal section (Fig 1) 3.2 Detailed analysis of cathepsin L colocalization with CTLA-2˛ protein in coronal sections from various regions of the mouse brain 3.2.1 Cerebral cortex and hippocampus Consistent double labeling for cathepsin L and CTLA-2␣ proteins was observed in the primary motor cortex, secondary motor cortex and somatosensory cortex High density of colocalization signals was detected in the corpus callosum (cc) which is a structure that connect the two hemispheres of the brain; the Cornu Ammonis 2, fields of hippocampus; alveus of the hippocampus (Alv); in neuron cell body of stratum pyramidale (Py) and fimbria of hippocampus(fi) (Fig 2a,b) Please cite this article in press as: Luziga, C., et al., Cathepsin L coexists with Cytotoxic T-lymphocyte Antigen-2 alpha in distinct regions of the mouse brain Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.08.003 G Model ACTHIS-51114; No of Pages ARTICLE IN PRESS C Luziga et al / Acta Histochemica xxx (2016) xxx–xxx Fig Double labeling immunofluorescence images showing Cathepsin L and CTLA-2␣ immunoreactivity in sagittal sections from various regions of the mouse brain (a) Demonstrates labeling for cathepsin L (green; FITC), (b) CTLA-2␣ (red; TRITC) and (c) merged image Very high immunoreactivity to indicate colocalization (yellow) in the merged image is seen in external capsule (ec), corpus callosum (gcc), fimbria of hippocampus (fi), Cornu Ammonis 2, fields of hippocampus (CA2 and CA3), stria medullaris of thalamus (sm) and mammillothalamic tract (mt) Moderate colocalization in isocortex, Lateral septal nucleus, dorsal part (LSD), Lateral septal nucleus, intermediate part (LSI), strial part of the preoptic area (StA) and anterodorsal thalamic nucleus (AD) but colocalization is not observed in molecular layer of hippocampus (Mol), dentate gyrus (DG), nucleus of the horizontal limb of the diagonal band (HDB), medial preoptic area (MPA) and medial preoptic nucleus, medial part (MPOM) Scale bar: 150 ␮m (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig (A) Double labeling immunofluorescence images showing Cathepsin L and CTLA-2␣ immunoreactivity in sagittal sections of the cortex and hippocampus The first column illustrates labeling for (a) cathepsin L (green; FITC), (b) CTLA-2␣ (red; TRITC) and (c) merged image in the subcortical structures and hippocampus Colocalization of Cathepsin L and CTLA-2␣ in the merged image is shown in corpus callosum (cc), Alveus of hippocampus (ALV) and Pyramidal cell layer of the hippocampus (Py) The second column demonstrates labeling for (d) cathepsin L (green; FITC), (e) CTLA-2␣ (red; TRITC) and (f) merged image in secondary motor cortex (M2), corpus callosum (cc), Cornu Ammonis field of hippocampus (CA2), fimbria of the hippocampus (fi), stria medullaris of thalamus (sm) and anterodorsal thalamic nucleus (AD) CTLA-2␣ immunoreactivity is observed in molecular layer of hippocampus (Mol) and dentate gyrus (DG), which does not colocalize with cathepsin L Scale bar: 70 ␮m (B) Immunofluorescence images demonstrating Cathepsin L and CTLA-2␣ double labeling immunoreactivity in coronal sections of the hippocampus The first column shows labeling for (a) cathepsin L (green; FITC), (b) CTLA-2␣ (red; TRITC) and (c) merged image in Cornu Ammonis 2, fields of hippocampus (CA2 and CA3) Strong immunoreactivity for cathepsin L is observed in CA2 and CA3 fields and colocalizes (yellow) with CTLA-2␣ in the merged image The second column illustrates labeling for (d) cathepsin L (green; FITC), (e) CTLA-2␣ (red; TRITC) and (f) merged image in the hippocampus Labeling for cathepsin L is seen in Cornu Ammonis field (CA3) and colocalizes with CTLA-2␣ (yellow) in the merged image but colocalization is not observed in molecular layer of hippocampus (Mol), dentate gyrus (DG) and in the hilus (h) Scale bar: 50 ␮m (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3.2.2 Amygdala The amygdala showed strong colocalization signals for cathesin L and CTLA-2␣ proteins in the capsular part (CeC) and lateral division (CeL) of central amygdaloid nucleus while at low level in the basolateral amygdaloid nucleus, anterior part (BLA) and absent in the negative control sections incubated with 10% normal serum in place of primary antibodies (Fig 3) 3.2.3 Ventricular system and thalamus Choroid plexus located in the ventricular system is important in maintaining generation and flow of cerebrospinal fluid (CSF) The plexus displayed high level of cathepsin L immunoreactivity colocalized with CTLA-2␣ within epindymal cells and medial habenular nucleus, a chief relay nucleus of the descending dorsal diencephalic conduction system The thalamic nuclei displayed dense to moderate level of colocalization for cathepsin L and CTLA2␣ proteins Intense labeling was detected in the stria medullaris of thalamus (sm) and anteromedial thalamic nucleus (AM); moderately in paraventricular thalamic nucleus, posterior part (PVP), paraventricular thalamic nucleus (PV) and interanteromedial thalamic nucleus (IAM) as well as in ventral reunions thalamic nucleus (VRe), paraventricular hypothalamic nucleus, lateral magnocellular Please cite this article in press as: Luziga, C., et al., Cathepsin L coexists with Cytotoxic T-lymphocyte Antigen-2 alpha in distinct regions of the mouse brain Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.08.003 G Model ACTHIS-51114; No of Pages ARTICLE IN PRESS C Luziga et al / Acta Histochemica xxx (2016) xxx–xxx Table Morphological structures refer to the neuron-anatomical atlas from Paxinos and Franklin (2001) Brain Region Density of positive cells Neocortex and subcortical regions Primary somatosensory cortex Secondary somatosensory cortex Retrosplenial cortex Secondary motor cortex Corpus callosum External capsule ++ ++ ++ ++ ++++ ++++ Septum region Fornix Lateral septal nucleus, dorsal part Lateral septal nucleus, intermediate part Nucleus of the anterior commissure ++++ ++ ++ +++ Amygdala BLA − Basolateral amygdaloid nucleus, anterior part CeC − Central amygdaloid nucleus, capsular part CeL − Central amygdaloid nucleus, lateral division + ++++ +++ Hippocampus Alveus of hippocampus Fimbria of hippocampus Dentate gyrus Cornu Ammonis 1, fields of hippocampus Cornu Ammonis 2, fields of hippocampus Cornu Ammonis,3 fields of hippocampus Hilus Dentate gyrus Molecular layer of hippocampus Stratum oriens Pyramidal cell layer of the hippocampus ++++ ++++ − − +++ ++++ − − − − ++++ Thalamus Stria medullaris of thalamus Anterodorsal thalamic nucleus Mediodorsal thalamic nucleus, central part Mammillothalamic tract External medullary lamina Medial habenular bodies Choroid plexus Anteromedial thalamic nucleus Paraventricular thalamic nucleus, posterior part Paraventricular thalamic nucleus Paraventricular thalamic nucleus, anterior part Interanteromedial thalamic nucleus Central medial thalamic nucleus Ventral reunions thalamic nucleus ++++ + +++ ++++ +++ +++ ++++ +++ +++ +++ − +++ + + Hypothalamus Strial part of the preoptic area Medial preoptic area Medial preoptic nucleus, medial part Septohypothalamic nucleus Paraventricular hypothalamic nucleus, posterior part Paraventricular hypothalamic nucleus, lateral magnocellular part Anterior hypothalamic area, anterior part Anterior hypothalamic area, posterior part Anterior hypothalamic area, central part Lateroanterior hypothalamic nucleus Posterior hypothalamic area Medial mammillary nucleus, medial part Medial mammillary nucleus, lateral part Supramammillary nucleus Interpeduncular nucleus, caudal subnucleus + + + + + + – + + +++ − +++ ++ +++ − Raphe nuclei (Midbrain) Paramedian raphe nucleus Dorsal raphe nucleus Median raphe nucleus ++ ++ ++ Cerebellum Molecular layer Purkinje cell layer Granule cell layer White matter + +++ +++ ++++ The intensity of cathepsin L colocalization with CTLA-2␣ was classified as follows: negative (−), moderate (++), high (+++), Very high (++++) The structures used to evaluate immunofluorescence colocalization were Dentate gyrus and Molecular layer of hippocampus (−); Central medial thalamic nucleus and Anterior hypothalamic area, posterior part (+); Lateral septal nucleus, dorsal part and Lateral septal nucleus, intermediate part (++); Anteromedial thalamic nucleus and Paraventricular thalamic nucleus (+++); Stria medullaris of thalamus and Mammillothalamic tract (++++) Fig Immunofluorescence images demonstrating Cathepsin L and CTLA-2␣ double labeling immunoreactivity in coronal sections of the amygdala The first column shows labeling for (a) cathepsin L (green; FITC), (b) CTLA-2␣ (red; TRITC) and (c) merged image Colocalization of Cathepsin L and CTLA-2␣ in the merged image is strong in capsular part (CeC) and lateral division (CeL) of central amygdaloid nucleus while at low level in the basolateral amygdaloid nucleus, anterior part (BLA) For control purposes, coronal sections from the amygdala were incubated with 10% normal serum in place of primary antibodies Labeling for cathepsin L and CTLA-2␣ is virtually absent in sections (d), (e) and (f) incubated with the 10% normal serum Scale bar: 50 ␮m (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) part (PaLM), and paraventricular hypothalamic nucleus, posterior part (PaPo) (Fig 4A and B) 3.2.4 Hypothalamus In the hypothalamus, strong immunoreactivity for cathepsin L colocalized with CTLA-2␣ was confined to the anterior commissure (ac), lateroanterior hypothalamic nucleus (LA); supramammillary nucleus (SuM); lateral part of medial mammillary nucleus (ML); moderately in anterior hypothalamic area, central part (AHC) and medial mammillary nucleus, medial part (MM) (Figs B and ) 3.2.5 Cerebellum In the cerebellum, intense double immunolabeling for cayhepsin L and CTLA-2␣ proteins was strong in the internal white matter; moderately in the granule layer in randomly distributed cells that represent Golgi cells and/or granule cells and in cell bodies of Purkinje neurons and low in the molecular layer in stellate and basket cells (Fig 6) Discussion Previous immunohistochemical studies show that CTLA-2␣ protein in the mouse brain is preferentially localized within dendrites and axonal fibres (Luziga et al., 2007) CTLA-2␣ is also shown to exhibit selective inhibition to mouse cathepsin L-like cysteine proteinases (Kurata et al., 2003) And that transient expression of crammer (cysteine proteinases inhibitor) correlates well with the establishment of long-term memory, suggesting a role of the crammer in memory formation through regulation of cathepsin activity (Comas et al., 2004) Dash et al (2000) also demonstrated that concurrent inhibition of multiple caspases (a family of cysteine proteases) in hippocampus blocks long-term but not short-term spatial memory in mouse brain In this context, we developed inter- Please cite this article in press as: Luziga, C., et al., Cathepsin L coexists with Cytotoxic T-lymphocyte Antigen-2 alpha in distinct regions of the mouse brain Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.08.003 G Model ACTHIS-51114; No of Pages ARTICLE IN PRESS C Luziga et al / Acta Histochemica xxx (2016) xxx–xxx Fig (A) Double labeling Immunofluorescence images showing Cathepsin L and CTLA-2␣ immunoreactivity in coronal sections of the Ventricular system and Thalamus The first column illustrates labeling for (a) cathepsin L (green; FITC), (b) CTLA-2␣ (red; TRITC) and (c) merged image in medial habenular nucleus (MHb) and Choroid plexus (chp) Very Strong labeling for both cathepsin L and CTLA-2␣ (yellow) is seen in medial habenular nucleus (MHb) and choroid plexus (chp) The second column shows labeling for (d) cathepsin L (green; FITC), (e) CTLA-2␣ (red; TRITC) and (f) merged image in the thalamus Very strong colocalization (yellow) is seen in stria medullaris of thalamus (sm) and anteromedial thalamic nucleus (AM) Scale bar: 70 ␮m (B) Double labeling immunofluorescence images demonstrating Cathepsin L and CTLA-2␣ immunoreactivity in sagittal sections of the thalamus The first column illustrates labeling for (a) cathepsin L (green; FITC), (b) CTLA-2␣ (red; TRITC) and (c) merged image in thalamus Moderate double labeling for both Cathepsin L and CTLA-2␣ (yellow) in the merged image is seen in paraventricular thalamic nucleus, posterior part (PVP), paraventricular thalamic nucleus (PV), interanteromedial thalamic nucleus (IAM) but is not observed in the anterior part of paraventricular thalamic nucleus, (PVA) The second column shows labeling for (d) cathepsin L (green; FITC), (e) CTLA-2␣ (red; TRITC) and (f) merged image in the thalamus Low intensity of colocalization (yellow) for both cathepsin L and CTLA-2␣ in the merged is seen in ventral reunions thalamic nucleus (VRe), paraventricular hypothalamic nucleus, lateral magnocellular part (PaLM), and paraventricular hypothalamic nucleus, posterior part (PaPo) Below the thalamus is the hypothalamus where moderate colocalization of cathepsin L and CTLA-2␣ is seen in the posterior part (AHP) but not in the anterior part of the anterior hypothalamic area (AHA) Scale bar: 100 ␮m (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) est to know the cellular colocalization of cathepsin L (a family of cystein proteases) with CTLA-2␣ protein in the mouse brain Understanding the cellular relationship of cathepsin inhibitory activity of CTLA-2␣ in light of the emerging roles of cathepsins in memory establishment, is essential in the development of treatments for degenerative diseases associated with learning and memory loss In this study, immunoreactivity for cathepsin L and CTLA-2␣ was detected in nerve fibres bundles and in some nerve cell bodies In the cerebral cortex and subcortical structures, colocalization was very high in neocortex and corpus callosum The neocortex is involved in higher brain functions such as sensory perception, generation of motor commands, spatial reasoning, conscious thought and language It has also an influential role in sleep, memory and learning processes (Lui et al., 2011) The corpus callosum plays a major role in most communications between different regions of the brain Studies show that cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides into active neuropeptides that are released to mediate cell to cell communication in the nervous system for neurotransmission (Hook et al., 2012) Identification of cathepsin L and CTLA-2␣ in neocortex and corpus callosum is suggestive of their regulatory role in increasing or decreasing information transfer in the brain and may also be involved in many other biological processes in the central nervous system that are yet to be identified The alveus, fimbria and the Pyramidal cell layer in Cornu Ammonis 2, fields of the hippocampus also showed intense double labeling for cathepsin L and CTLA-2␣ The alveus is composed of the white myelinated fibres that arise from cell bodies of subculum and hippocampus and eventually merges with the fimbria of the hippocampus that goes on to become the fornix, which is also a prominent white matter tracts passing above the thalamus Fibres of the fornix travel to the anterior commissure, a white matter tract connecting both hemispheres and terminate in the mammillary bodies and continue upward through the mammilothalamic tract towards the anterior nucleus of the thalamus (Maren, 1999; Amunts et al., 2005; Marc and Sergio, 2014) All these structures are part of the limbic system including amygdala, habenular and raphae nucleus and were strongly labelled for cathepsin L and CTLA-2␣ The presence of cathepsin L and CTLA-2␣ in these structures is suggestive of their significant role in regulation of limbic system functions that include motivation and reward, emotion, learning, and memory establishment One of the most prominent labeling structures for both cathepsin L and CTLA-2␣ was the choroid plexus epithelium that produces and secretes cerebrospinal fluid and controls movement of solutes between the blood and the cerebrospinal fluid The fluid provides mechanical protection and a stable physiological environment for the central nervous system and supplies the brain with certain nutrients, hormones, and metal ions, while clearing cells that populate the anatomical compartment (Purves et al., 2001) Localization of cathespin L and CTLA-2␣ in these structures correlated well with our previous studies on the distribution of both cathespin L and CTLA-2␣ in choroid plexus epithelium (Luziga et al., 2008, 2016) Whether the colocalization of cathespin L and CTLA-2␣ to choroid plexus epithelium is related to its role to produce and secrete cerebrospinal fluid; regulate and transfer of molecules across the bloodcerebrospinal fluid interface or to any other novel function is a question that remains to be resolved The stria medullaris, mammillothalamic tract, anterodorsal thalamic nucleus and anteromedial thalamic nucleus in thalamus displayed strong double immunofluorescence labeling for cathepsin L and CTLA-2␣ In functional perspective, the anterior nuclei Please cite this article in press as: Luziga, C., et al., Cathepsin L coexists with Cytotoxic T-lymphocyte Antigen-2 alpha in distinct regions of the mouse brain Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.08.003 G Model ACTHIS-51114; No of Pages ARTICLE IN PRESS C Luziga et al / Acta Histochemica xxx (2016) xxx–xxx Fig Double labeling immunofluorescence images showing Cathepsin L and CTLA2␣ immunoreactivity in sagittal sections of the hypothalamus The first column shows labeling for (a) cathepsin L (green; FITC), (b) CTLA-2␣ (red; TRITC) and (c) merged image in hypothalamus Intense immunoreactivity of cathepsin L is observed in lateroanterior hypothalamic nucleus (LA) and colocalizes with CTLA-2␣ in the merged imerge Moderate colocalization is observed in anterior hypothalamic area, central part (AHC) The second column illustrates labeling for (d) cathepsin L (green; FITC), (e) CTLA-2␣ (red; TRITC) and (f) merged image in the hypothalamus Strong double labeling for cathepsin L and CTLA-2␣ is evident in supramammillary nucleus (SuM) and lateral part of medial mammillary nucleus (ML); moderately in medial mammillary nucleus, medial part (MM) but low in paramedian raphe nucleus (PMnR) and absent in interpeduncular nucleus, caudal subnucleus (IPL) Scale bar: 100 ␮m (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) and the mediodorsal thalamic nuclei act as nodal points of convergence where bilateral tissue damage can result in memory function impairment similar to bilateral lesions of the mammillothalamic tract, which connects the mammillary bodies to the anterior nuclei (Berger, 2004) In rodents, bilateral disruption of this pathway has also been shown to attenuate reinstatement of drug seeking behavior (Suzanne et al., 2001) Whether the colocalization of cathepsin L and CTLA-2␣ to these structures is related to role in establishment of recall memory or to a novel function is a matter that needs further studies In the cerebellum, abundant double labeling signals for cathepsin L and CTLA-2␣ were distributed in the white matter, granules cell layer and Purkinje cells and moderately in the molecular layer The Purkinje cells are the only neurons responsible for sending output from the cortex On the other hand cathepsin L and CTLA2␣ were locacalized in deep cerebellar nuclei in the white matter which receives input from the Purkinje cells and send output information to other brain regions Studies show that Purkinje cells are affected by the combined loss of cathepsin B and L while overexpression of the cathepsins contributes to neurodegeneration of granule cells (Sevenich et al., 2006) Presence of cathepsin L and CTLA-2␣ in the cerebellum is suggestive of their regulatory role in prevention of Purkinje and granule cell degeneration In conclusion, this study shows that cathepsin L coexists with CTLA-2␣ in distinct regions of the mouse brain including cerebral cortex, subcortical structures, hippocampus, amygdala, thalamic nuclei, hypothalamus and the cerebellum The colocalization indicate distinct structural or regional specific function of cathespin L and CTLA-2␣ in various physiological and pathological processes of the nervous system and that the fine equilibrium between the syn- Fig Immunofluorescence images demonstrating Cathepsin L and CTLA-2␣ immunoreactivity double labeling in sagittal sections of the cerebellum The first column illustrates labeling for (a) cathepsin L (green; FITC), (b) CTLA-2␣ (red; TRITC) and (c) merged image in cerebellum Colocalization (yellow); for cathepsin L and CTLA-2␣ in the merged image is strong in the internal white matter; moderately in the granule layer in randomly distributed cells that represent Golgi cells and/or granule cells and in cell bodies of Purkinje neurons and low in the molecular layer in stellate and basket cells The second column shows larger magnification images labelled for (d) cathepsin L (green; FITC), (e) CTLA-2␣ (red; TRITC) and (f) merged in the cerebellum Scale bar: a, b, c: 100 ␮m; d, e, f: 50 ␮m (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) thesis and secretion of cathespin L and CTLA-2␣ is part of the brain processes for establishment of long-term memory, storage of memory traces for spatial information, olfaction, emotion, behavior and motivation and hence maintaining normal growth and development This study also opens ways to new studies on the functional implications of cathepsin L and CTLA-2␣ in the central nervous system Acknowledgement The authors 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activity of CTLA -2 in light... paraventricular hypothalamic nucleus, lateral magnocellular Please cite this article in press as: Luziga, C., et al., Cathepsin L coexists with Cytotoxic T- lymphocyte Antigen- 2 alpha in distinct regions. .. Please cite this article in press as: Luziga, C., et al., Cathepsin L coexists with Cytotoxic T- lymphocyte Antigen- 2 alpha in distinct regions of the mouse brain Acta Histochemica (20 16), http://dx.doi.org/10.1016/j.acthis .20 16.08.003