Studying the Effect of Lactobacillus fermentum on Arc and CREB Genes Expression of Involved in The Memory of Alzheimer\'s Rats

Introduction: Alzheimer's disease is a common progressive neurogenerative disease that leads to dementia and destruction of brain cells, especially in areas related to learning and memory such as the hippocampus. The role of the Arc gene in synaptic flexibility and memory consolidation has been proven and its expression is strongly influenced by neuronal activity. The aim of this study was to investigate the effect of Lactobacillus fermentum on the expression of Arc (effective in synaptic flexibility and memory consolidation) and CREB (effective in stabilizing synaptic changes during learning) genes, involved in the memory of Alzheimer's rats. Material & ethods: In this study that was conducted in 2019, 30 male Wistar rats were divided into 5 groups (control, Alzheimer and 3 groups as treatment). In the treatment groups, simultaneously with induction of Alzheimer by stereotaxic method with streptozotocin, supernatant of Lactobacillus fermentum PTCC 1744 in MRS broth with doses of 108, 107, and 106 cfu/ml was injected intraperitoneally for 21 days. After RNA extraction from hippocampus samples, cDNA was synthesized and the expression of the genes was evaluated by real-time PCR and LinReg PCR software. Findings: The data showed an increase in gene expression of Arc and CREB in the treatment groups with a dose of 106 cfu/ml compared to the Alzheimer's group and the difference was significant (P <0.001). Discussion & conclusion: Extracellular compounds of L. fermentum may inhibit the progression of neuronal lesions due to their antioxidant and anti-inflammatory effects and may be effective in improving Alzheimer's

Fractalkine enhances oligodendrocyte regeneration and remyelination in a demyelination mouse model

Demyelinating disorders of the central nervous system (CNS) occur when myelin and oligodendrocytes are damaged or lost. Remyelination and regeneration of oligodendrocytes can be achieved from endogenous oligodendrocyte precursor cells (OPCs) that reside in the adult CNS tissue. Using a cuprizone mouse model of demyelination, we show that infusion of fractalkine (CX3CL1) into the demyelinated murine brain increases de novo oligodendrocyte formation and enhances remyelination in the corpus callosum and cortical gray matter. This is achieved by increased OPC proliferation in the cortical gray matter as well as OPC differentiation and attenuation of microglia/macrophage activation both in corpus callosum and cortical gray matter. Finally, we show that activated OPCs and microglia/macrophages express fractalkine receptor CX3CR1 in vivo, and that in OPC-microglia co-cultures fractalkine increases in vitro oligodendrocyte differentiation by modulating both OPC and microglia biology. Our results demonstrate a novel pro-regenerative role of fractalkine in a demyelinating mouse model

Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments

List of primers and their sequences or catalogue numbers used in the study.

<p>List of primers and their sequences or catalogue numbers used in the study.</p

Characterization of HUVEC-derived iPSCs (HiPSCs).

<p>Gene expression was determined after 5 passages of the colonies. RT-PCR results (mRNA levels) showing endogenous embryonic (A) and endothelial (B) specific genes expressed by HUVECs and HiPSCs. Values in Figure B represent fold reduction compared to HUVECs. Data are average values of 4 HiPSC lines (N = at least 3 replicates; P<0.001). Non-detectable genes are denoted as n.d. At protein levels using immune-staining, representative micrographs show expression of embryonic markers-SSEA-4 and Oct 3/4 (C and D respectively), DAPI (E) and lack of expression of endothelial markers- PECAM (CD31) and VE-cadherin (F and G respectively) in HiPSC colonies. H is DAPI co-staining of panels F and G. Scale bar: 200 μm.</p

Calcium imaging and expression of functional receptors on neurons.

<p>(A) Primary human fetal neurons (HFNs) and (B) HiPSC-derived neurons (HiPSC-Ns) were incubated with the membrane-permeant acetoxymethyl form of the fluorescent Ca²⁺-sensitive dye Fluo-4-AM for 1hr prior to imaging, after which the fluorescence intensity was measured. Graphs shown on left column represent responses of three HFNs, and on right responses of three HiPSC-Ns to glutamate, nicotine and acetylcholine treatments. Arrows indicate the time when the stimulants were applied. Scale bar: 100 μm. The experiments were repeated 3 times.</p

Calcium imaging and expression of functional receptors on astrocytes.

<p>(A) Primary human fetal astrocytes (HFAs) and (B) HiPSC-derived astrocytes (HiPSC-As) were incubated with the membrane-permeant acetoxymethyl form of the fluorescent Ca²⁺-sensitive dye Fluo-4-AM for 1hr prior to imaging, after which fluorescence intensity was measured. Graphs shown on left column represent responses of three HFAs and on right responses of three HiPSC-As to ATP and acetylcholine treatments. Arrows indicate the time when the stimulants were applied. Scale bar: 100 μm. The experiments were repeated 3 times.</p