The cerebellum is involved in higher order cognitive function and is susceptible to age-related atrophy. However, limited evidence has directly examined the cerebellum\'s role in cognitive aging. To interrogate potential substrates of the relationship between cerebellar structure and memory in aging, here we target the Purkinje cells (PCs). The sole output neurons of the cerebellum, PC loss and/or degeneration underlie a variety of behavioral abnormalities. Using a rat model of normal cognitive aging, we immunostained sections through the cerebellum for the PC-specific protein, calbindin-D28k. Although morphometric quantification revealed no significant difference in total PC number as a function of age or cognitive status, regional cell number was a more robust correlate of memory performance in the young cerebellum than in aged animals. Parallel biochemical analysis of PC-specific protein levels in whole cerebellum additionally revealed that calbindin-D28k and Purkinje cell protein-2 (pcp-2) levels were lower selectively in aged rats with spatial memory impairment compared to both young animals and aged rats with intact memory. These results suggest that cognitive aging is associated with cerebellum vulnerability, potentially reflecting disruption of the cerebellum-medial temporal lobe network.

OBJECTIVE: This study aims to investigate the effect of calbindin 1 on the proliferation and apoptosis of gingival epithelial cells affected by Porphyromonas gingivalis (P. gingivalis)invitro.
METHODS: A model of P. gingivalis infecting CA9-22 was established in vitro. At 24 h after infection, the expression of calbindin 1 (CALB1) was detected by real-time fluorescent quantitative polymerase chain reaction, Western blot, and immunofluorescence analyses. The expression of CALB1 was further inhibited by RNA interference. Cell proliferation was detected by BrdU analysis, and cell apoptosis was detected by caspase 3 activity. The expression of MDM2 and p53 was detected by Western blot analysis.
RESULTS: P. gingivalis infection upregulated the expression of CALB1 in CA9-22 cells with multiplicity-dependent manner. CALB1 promoted the proliferation of CA9-22 cells, increased the expression of MDM2, and inhibited the expression of p53. Inhibiting CALB1 expression did not affect the inhibitory effect of P. gingivalis infection on CA9-22 apoptosis.
CONCLUSIONS: P. gingivalis infection can promote the proliferation of CA9-22 cells by increasing CALB1 expression. The related mechanism may be associated with MDM2-p53.
目的: 探究钙结合蛋白1在牙龈卟啉单胞菌（P. gingivalis）影响牙龈上皮细胞增殖和凋亡中的作用。方法: P. gingivalis感染CA9-22细胞，在感染24 h后，采用实时荧光定量聚合酶链反应、免疫印迹法和免疫荧光法检测钙结合蛋白1（CALB1）的表达。通过RNA干扰法抑制CALB1表达，BrdU分析检测细胞增殖，Caspase 3活性测定检测细胞凋亡，免疫印迹法检测MDM2和p53的表达。结果: P. gingivalis感染促进CA9-22细胞中CALB1的表达，并且CALB1的表达具有感染复数依赖性。CALB1促进CA9-22细胞增殖，上调细胞内MDM2的表达，同时抑制p53的表达。下调CALB1表达不影响P. gingivalis感染对CA9-22凋亡的抑制作用。结论: P. gingivalis感染可通过上调CALB1表达促进CA9-22细胞增殖，其机制可能与影响MDM2-p53有关。.

Amyloid β (Aβ) oligomers are the most neurotoxic forms of Aβ, and Aβ(1-42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer\'s disease patients. Aβ(25-35) is the shortest peptide that retains the toxicity of Aβ(1-42). Aβ oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aβ(1-42) concentration range. Aβ and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aβ(1-42). The formation of His6/Aβ(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aβ(1-42) and Aβ(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets.

The inhibitory neurons in the brain play an essential role in neural network firing patterns by releasing γ-aminobutyric acid (GABA) as the neurotransmitter. In the mouse brain, based on the protein molecular markers, inhibitory neurons are usually to be divided into three non-overlapping groups: parvalbumin (PV), neuropeptide somatostatin (SST), and vasoactive intestinal peptide (VIP)-expressing neurons. Each neuronal group exhibited unique properties in molecule, electrophysiology, circuitry, and function. Calbindin 1 (Calb1), a ubiquitous calcium-binding protein, often acts as a \"divider\" in excitatory neuronal classification. Based on Calb1 expression, the excitatory neurons from the same brain region can be classified into two subgroups with distinct properties. Besides excitatory neurons, Calb1 also expresses in part of inhibitory neurons. But, to date, little research focused on the intersectional relationship between inhibitory neuronal subtypes and Calb1. In this study, we genetically targeted Calb1-expression (Calb1+) and Calb1-lacking (Calb1-) subgroups of PV and SST neurons throughout the mouse brain by flexibly crossing transgenic mice relying on multi-recombinant systems, and the distribution patterns and electrophysiological properties of each subgroup were further demonstrated. Thus, this study provided novel insights and strategies into inhibitory neuronal classification.

BACKGROUND: While most animals of the Muridae family are nocturnal, the gerbil displays diurnal activity and provides a useful model for visual system research. The purpose of this study was to investigate the localization of calcium-binding proteins (CBPs) in the visual cortex of the Mongolian gerbil (Meriones unguiculatus). We also compared the labeling of CBPs to those of gamma-aminobutyric acid (GABA)- and nitric oxide synthase (NOS)-containing neurons.
METHODS: The study was conducted on twelve adult Mongolian gerbils (3-4 months old). We used horseradish peroxidase immunocytochemistry and two-color fluorescence immunocytochemistry with conventional and confocal microscopy to assess CBPs localization in the visual cortex.
RESULTS: The highest density of calbindin-D28K (CB)- (34.18%) and parvalbumin (PV)-IR (37.51%) neurons was found in layer V, while the highest density of calretinin (CR)-IR (33.85%) neurons was found in layer II. The CB- (46.99%), CR- (44.88%), and PV-IR (50.17%) neurons mainly displayed a multipolar round/oval morphology. Two-color immunofluorescence revealed that only 16.67%, 14.16%, and 39.91% of the CB-, CR-, and PV-IR neurons, respectively, contained GABA. In addition, none of the CB-, CR-, and PV-IR neurons contained NOS.
CONCLUSIONS: Our findings indicate that CB-, CR-, and PV-containing neurons in the Mongolian gerbil visual cortex are distributed abundantly and distinctively in specific layers and in a small population of GABAergic neurons but are limited to subpopulations that do not express NOS. These data provide a basis for the potential roles of CBP-containing neurons in the gerbil visual cortex.

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by congenital anomalies, cancer predisposition and a severe hypo-proliferative anemia. It was the first disease linked to ribosomal dysfunction and >70 % of patients have been identified to have a haploinsufficiency of a ribosomal protein (RP) gene, with RPS19 being the most common mutation. There is significant variability within the disease in terms of phenotype as well as response to therapy suggesting that other genes contribute to the pathophysiology and potential management of this disease. To explore these questions, we performed a genome-wide CRISPR screen in a cellular model of DBA and identified Calbindin 1 (CALB1), a member of the calcium-binding superfamily, as a potential modifier of the disordered erythropoiesis in DBA. We used human derived CD34+ cells cultured in erythroid stimulating media with knockdown of RPS19 as a model for DBA to study the effects of CALB1. We found that knockdown of CALB1 in this DBA model promoted erythroid maturation. We also noted effects of CALB1 knockdown on cell cycle. Taken together, our results reveal CALB1 is a novel regulator of human erythropoiesis and has implications for using CALB1 as a novel therapeutic target in DBA.

Rabies virus (RABV) infection leads to a fatal neurological outcome in humans and animals and is associated with major alterations in cellular gene expression. In this study, we describe the effects of RABV infection on the mRNA expression levels of two genes, encoding the Ca2+-binding proteins (Ca-BPs) calbindin D-28K (Calb1) and calretinin (Calb2), in the brains of BALB/c mice. Sixty 4-week-old mice were divided into two test groups and one control group. Mice were inoculated intramuscularly with either a street rabies virus (SRV) strain or a challenge virus standard (CVS-11) strain and sacrificed at 3-day intervals up to day 18 postinfection. A direct fluorescent antibody test (DFAT) was used to verify the presence of RABV antigen in brain tissues, and real-time quantitative PCR (RT-PCR) was used to assess gene expression. Infection with both RABV strains resulted in significant (p < 0.05) increases in Calb1 and Calb2 expression in the test animals when compared with the controls at various time points in the study. Correlation analysis indicated very weak insignificant (p > 0.05) negative and positive relationships, respectively, between Calb1 expression (r = -0.04) and Calb2 expression (r = 0.08) with viral load (CVS-11 strain). Insignificant (p > 0.05) relationships were also observed Calb1 expression (r = -0.28) and Calb2 expression (r = 0.06) and viral load for the SRV strain.The observed alterations in Calb1 and Calb2 expression in this study indicate possible impairments in neuronal Ca2+ buffering and Ca2+ homeostasis as a result of RABV infection and, consequently, possible involvement of calbindin-D28K and calretinin in the neuropathogenesis of rabies.

The incidence rates of light-induced retinopathies have increased significantly in the last decades because of continuous exposure to light from different electronic devices. Recent studies showed that exposure to blue light had been related to the pathogenesis of light-induced retinopathies. However, the pathophysiological mechanisms underlying changes induced by light exposure are not fully known yet. In the present study, the effects of exposure to light at different wavelengths with emission peaks in the blue light range (400-500 nm) on the localization of Calretinin-N18 (CaR-N18) and Calbindin-D28K (CaB-D28K) in adult zebrafish retina are studied using double immunofluorescence with confocal laser microscopy. CaB-D28K and CaR-N18 are two homologous cytosolic calcium-binding proteins (CaBPs) implicated in essential process regulation in central and peripheral nervous systems. CaB-D28K and CaR-N18 distributions are investigated to elucidate their potential role in maintaining retinal homeostasis under distinct light conditions and darkness. The results showed that light influences CaB-D28K and CaR-N18 distribution in the retina of adult zebrafish, suggesting that these CaBPs could be involved in the pathophysiology of retinal damage induced by the short-wavelength visible light spectrum.

Recent clinical and experimental studies have highlighted the involvement of Ventral Tegmental Area (VTA) dopamine (DA) neurons for the early pathogenesis of Alzheimer\'s Disease (AD). We have previously described a progressive and selective degeneration of these neurons in the Tg2576 mouse model of AD, long before amyloid-beta plaque formation. The degenerative process in DA neurons is associated with an autophagy flux impairment, whose rescue can prevent neuronal loss. Impairments in autophagy can be the basis for accumulation of damaged mitochondria, leading to disturbance in calcium (Ca2+) homeostasis, and to functional and structural deterioration of DA neurons.
In Tg2576 mice, we performed amperometric recordings of DA levels and analysis of dopaminergic fibers in the Nucleus Accumbens - a major component of the ventral striatum precociously affected in AD patients - together with retrograde tracing, to identify the most vulnerable DA neuron subpopulations in the VTA. Then, we focused on these neurons to analyze mitochondrial integrity and Apoptosis-inducing factor (AIF) localization by electron and confocal microscopy, respectively. Stereological cell count was also used to evaluate degeneration of DA neuron subpopulations containing the Ca2+-binding proteins Calbindin-D28K and Calretinin. The expression levels for these proteins were analyzed by western blot and confocal microscopy. Lastly, using electrophysiology and microfluorometry we analyzed VTA DA neuron intrinsic properties and cytosolic free Ca2+ levels.
We found a progressive degeneration of mesolimbic DA neurons projecting to the ventral striatum, located in the paranigral nucleus and parabrachial pigmented subnucleus of the VTA. At the onset of degeneration (3 months of age), the vulnerable DA neurons in the Tg2576 accumulate damaged mitochondria, while AIF translocates from the mitochondria to the nucleus. Although we describe an age-dependent loss of the DA neurons expressing Calbindin-D28K or Calretinin, we observed that the remaining cells upregulate the levels of Ca2+-binding proteins, and the free cytosolic levels of Ca2+ in these neurons are significantly decreased. Coherently, TUNEL-stained Tg2576 DA neurons express lower levels of Calbindin-D28K when compared with non-apoptotic cells.
Overall, our results suggest that the overexpression of Ca2+-binding proteins in VTA DA neurons might be an attempt of cells to survive by increasing their ability to buffer free Ca2+. Exploring strategies to overexpress Ca2+-binding proteins could be fundamental to reduce neuronal suffering and improve cognitive and non-cognitive functions in AD.

The scientific research community devotes stupendous efforts to control the arguable counterbalance between the undesirable effects of hormone replacement therapy (HRT) and post-menopausal syndrome. The recent emergence of 3rd generation selective estrogen receptor modulators and phytoestrogens has provided a promising alternative to HRT. Hence, we assessed the potential effects of combined Bazedoxifene and Genistein on hippocampal neuro-alterations induced by experimental ovariectomy.
For this purpose, we utilized forty-eight healthy sexually mature female Wistar rats assorted to control, ovariectomy (OVX), Genistein-treated ovariectomized (OVX+GEN) and Bazedoxifene and Genistein-treated ovariectomized (OVX+BZA+GEN) groups. Hippocampi samples from various groups were examined by H&E, silver stains and immunohistochemical examination for calbindin-D28k, GFAP, and BAX proteins. We also assessed hippocampal mRNA expression of ERK, CREB, BDNF and TrkB.
Our histopathological results confirmed that combined BZA+GEN induced restoration of hippocampal neuronal architecture, significant reduction of GFAP and BAX mean area % and significant upregulation of calbindin-D28k immunoexpression. Furthermore, we observed significant upregulation of ERK, CREB, BDNF and TrkB mRNA expression in the BZA+GEN group compared to the OVX group.
Taken together, our findings have provided a comprehensive assessment of histological, immunohistochemical and cyto-molecular basis of combined Genistein and Bazedoxifene ameliorative impacts on hippocampal neuro-alterations of OVX rats via upregulation of Calbindin, CERB, BDNF, Trk-B and ERK neuronal expression.