Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs

Branaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects; we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMN▵7), and in juvenile Wistar Hanover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.

Anatomical organization of the lateral cervical nucleus in Artiodactyls

The presence of the lateral cervical nucleus (LCN) in different mammals, including humans, has been established in a number of anatomical research works. The LCN receives its afferent inputs from the spinocervical tract, and conveys this somatosensory information to the various brain areas, especially the thalamus. In the present study, the organization of the calf and pig LCN was examined through the use of thionine staining and immunohistochemical methods combined with morphometrical analyses. Specifically, the localization of calbindin-D28k (CB-D28k) and neuronal nitric oxide synthase (nNOS) in the LCN was investigated using the immunoperoxidase method. Calf and pig LCN appear as a clearly defined column of gray matter located in the three cranial segments of the cervical spinal cord. Thionine staining shows that polygonal neurons represent the main cell type in both species. The calf and pig LCN contained CB-D28k-immunoreactive (IR) neurons of varying sizes. Large neurons are probably involved in the generation of the cervicothalamic pathway. Small CB-D28k-IR neurons, on the other hand, could act as local interneurons. The immunoreactivity for nNOS was found to be mainly located in thin neuronal processes that could represent the terminal axonal portion of nNOS-IR found in laminae III e IV. This evidence suggests that nitric oxide (NO) could modulate the synaptic activity of the glutamatergic spinocervical tracts. These findings suggest that the LCN of Artiodactyls might play an important role in the transmission of somatosensory information from the spinal cord to the higher centers of the brain.

Anatomical Organization of the Lateral Cervical Nucleus in Artiodactyls

The presence of the lateral cervical nucleus (LCN) in different mammals, including humans, has been established in a number of anatomical research works. The LCN receives its afferent inputs from the spinocervical tract, and conveys this somatosensory information to the various brain areas, especially the thalamus. In the present study, the organization of the calf and pig LCN was examined through the use of thionine staining and immunohistochemical methods combined with morphometrical analyses. Specifically, the localization of calbindin-D28k (CB-D28k) and neuronal nitric oxide synthase (nNOS) in the LCN was investigated using the immunoperoxidase method. Calf and pig LCN appear as a clearly defined column of gray matter located in the three cranial segments of the cervical spinal cord. Thionine staining shows that polygonal neurons represent the main cell type in both species. The calf and pig LCN contained CB-D28k-immunoreactive (IR) neurons of varying sizes. Large neurons are probably involved in the generation of the cervicothalamic pathway. Small CB-D28k-IR neurons, on the other hand, could act as local interneurons. The immunoreactivity for nNOS was found to be mainly located in thin neuronal processes that could represent the terminal axonal portion of nNOS-IR found in laminae III e IV. This evidence suggests that nitric oxide (NO) could modulate the synaptic activity of the glutamatergic spinocervical tracts. These findings suggest that the LCN of Artiodactyls might play an important role in the transmission of somatosensory information from the spinal cord to the higher centers of the brain.

Organization of Neuropeptide Y-Immunoreactive Cells in the Mongolian gerbil (Meriones unguiculatus) Visual Cortex

Neuropeptide Y (NPY) is found throughout the central nervous system where it appears to be involved in the regulation of a wide range of physiological effects. The Mongolian gerbil, a member of the rodent family Muridae, is a diurnal animal and has been widely used in various aspects of biomedical research. This study was conducted to investigate the organization of NPY-immunoreactive (IR) neurons in the gerbil visual cortex using NPY immunocytochemistry. The highest density of NPY-IR neurons was located in layer V (50.58%). The major type of NPY-IR neuron was a multipolar round/oval cell type (44.57%). Double-color immunofluorescence revealed that 89.55% and 89.95% of NPY-IR neurons contained gamma-aminobutyric acid (GABA) or somatostatin, respectively. Several processes of the NPY-IR neurons surrounded GABAergic interneurons. Although 30.81% of the NPY-IR neurons contained calretinin, NPY and calbindin-D28K-IR neurons were co-expressed rarely (3.75%) and NPY did not co-express parvalbumin. Triple-color immunofluorescence with anti-GluR2 or CaMKII antibodies suggested that some non-GABAergic NPY-IR neurons may make excitatory synaptic contacts. This study indicates that NPY-IR neurons have a notable architecture and are unique subpopulations of the interneurons of the gerbil visual cortex, which could provide additional valuable data for elucidating the role of NPY in the visual process in diurnal animals.

Methionine supplementing effects on intestine, liver and uterus morphology, and on positivity and expression of Calbindin-D28k and TRPV6 epithelial calcium carriers in laying quail in thermoneutral conditions and under thermal stress

This study aimed to provide the performance, localization and expression of the epithelial calcium transporter channels Calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quail under heat stress (HS), and with methionine supplementation (MS). This study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels in the kidneys, intestine and uterus of 504 laying quails under different MS (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under HS (32°C) had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb, and increased positivity in proximal CTs. Deleterious effects of HS were minimized with MS for: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney (P≤0.05). Epithelial calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the MS, increasing egg production in HS and quality in termoneutrality (P≤0.05). MS further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity of Calb, and increase Calb and TRPV6 expression in the kidney (P≤0.001) under thermoneutrality. It was concluded that the use of MS (120%) is justifiable in order to partially reverse the deleterious effects of HS on the production, in the epithelial calcium carriers, and in the digestory and reproductive morphology of laying quail.

Increased Calbindin D28k Expression via Long-Term Alternate-Day Fasting Does Not Protect against Ischemia-Reperfusion Injury: A Focus on Delayed Neuronal Death, Gliosis and Immunoglobulin G Leakage

Calbindin-D28k (CB), a calcium-binding protein, mediates diverse neuronal functions. In this study, adult gerbils were fed a normal diet (ND) or exposed to intermittent fasting (IF) for three months, and were randomly assigned to sham or ischemia operated groups. Ischemic injury was induced by transient forebrain ischemia for 5 min. Short-term memory was examined via passive avoidance test. CB expression was investigated in the Cornu Ammonis 1 (CA1) region of the hippocampus via western blot analysis and immunohistochemistry. Finally, histological analysis was used to assess neuroprotection and gliosis (microgliosis and astrogliosis) in the CA1 region. Short-term memory did not vary significantly between ischemic gerbils with IF and those exposed to ND. CB expression was increased significantly in the CA1 pyramidal neurons of ischemic gerbils with IF compared with that of gerbils fed ND. However, the CB expression was significantly decreased in ischemic gerbils with IF, similarly to that of ischemic gerbils exposed to ND. The CA1 pyramidal neurons were not protected from ischemic injury in both groups, and gliosis (astrogliosis and microgliosis) was gradually increased with time after ischemia. In addition, immunoglobulin G was leaked into the CA1 parenchyma from blood vessels and gradually increased with time after ischemic insult in both groups. Taken together, our study suggests that IF for three months increases CB expression in hippocampal CA1 pyramidal neurons; however, the CA1 pyramidal neurons are not protected from transient forebrain ischemia. This failure in neuroprotection may be attributed to disruption of the blood–brain barrier, which triggers gliosis after ischemic insults.

IMMUNOHISTOCHEMICAL STUDY OF CALBINDIN IN THE DEVELOPING CEREBELLUM OF THE RAT

Background. Calbindin is a calcium-binding protein that supports calcium homeostasis for the normal functioning of neurons. Purpose. To study the distribution of immunoreactivity of calbindin-D28K in the structures of the developing cerebellum of the rat.Material and methods. The study was performed on 16 outbred white rats of different age groups: 2-, 7-, 15-days (early postnatal period), 45-days (puberty period). The cerebellum samples were taken and fixed in zinc-ethanol-formaldehyde for immunohistochemistry. Calbindin-D28K immunoreactivity was determined on paraffin sections using primary polyclonal rabbit antibodies.Results. In the cerebellar cortex, calbindin immunoreactivity was detected on the 2nd day after development of Purkinje cells (PC) in their perikaryons, and by the 15th day in their dendrites and it did not change by the 45th day. In all terms of the study in PC, it was detected not only in the cytoplasm, but also in their nucleus. In the granular layer, calbindin immunoreactivity decreased in rats in postnatal ontogenesis, however, in adult rats, some neurons were moderately immunopositive. Among them, from the 15th day after birth, the calbindin-immunoreactive afferent nerve fibers running in the white matter were detected. There were no significant differences in the distribution of calbindin between the lobes of the cerebellum of different phylogenetic age. Conclusions. Considering that the expression of сalbindin-D28k is detected throughout the entire period of development of Purkinje cells, as well as its physiological role in maintaining the function and homeostasis of calcium in them, it can be concluded that сalbindin-D28k is a valuable marker for the morphofunctional characteristics of PC in the developing and adult cerebellum of rats in normal and pathological conditions.

A Comprehensive Review of Alzheimer’s Association with Related Proteins: Pathological Role and Therapeutic Significance

: Alzheimer’s is an insidious, progressive, chronic neurodegenerative disease which causes the devastation of neurons. Alzheimer's possesses complex pathologies of heterogeneous nature counting proteins as one major factor along with enzymes and mutated genes. Proteins such as amyloid precursor protein (APP), apolipoprotein E (ApoE), presenilin, mortalin, calbindin-D28K, creactive protein, heat shock proteins (HSPs), and prion protein are some of the chief elements in the foremost hypotheses of AD like amyloid-beta (Aβ) cascade hypothesis, tau hypothesis, cholinergic neuron damage, etc. Disturbed expression of these proteins results in synaptic dysfunction, cognitive impairment, memory loss, and neuronal degradation. On the therapeutic ground, attempts of developing anti-amyloid, anti-inflammatory, anti-tau therapies are on peak, having APP and tau as putative targets. Some proteins, e.g., HSPs, which ameliorate oxidative stress, calpains, which help in regulating synaptic plasticity, and calmodulin-like skin protein (CLSP) with its neuroprotective role are few promising future targets for developing anti-AD therapies. On diagnostic grounds of AD C-reactive protein, pentraxins, collapsin response mediator protein-2, and growth-associated protein-43 represent the future of new possible biomarkers for diagnosing AD. The last few decades were concentrated over identifying and studying protein targets of AD. Here, we reviewed the physiological/pathological roles and therapeutic significance of nearly all the proteins associated with AD that addresses putative as well as probable targets for developing effective anti-AD therapies.