High-Intensity Interval Exercise Regulates Neurotrophic Factors and Astrocyte Activity in the Hippocampus and Cerebral Cortex

Parkinson disease is characterized by the selective degeneration of dopaminergic (DA) neurons in substantia nigra. Long term epidemiological studies have implicated exposure to agricultural pesticides as a significant risk factor. Systemic administration of rotenone, a widely used pesticide, causes selective degeneration of nigral DA neurons and Parkinson disease-like symptoms in rats. Our previous study has shown that the microtubule depolymerizing activity of rotenone plays a critical role in its selective toxicity on DA neurons. Rotenone toxicity is mimicked by the microtubule-depolymerizing drug colchicine and attenuated by the microtubule-stabilizing agent taxol. Here we show that nerve growth factor (NGF) significantly reduced rotenone toxicity on TH(+) neurons in midbrain neuronal cultures. The protective effect of NGF was completely abolished by inhibiting the microtubule-associated protein kinase kinase (MEK) and partially reversed by blocking phosphatidylinositol 3-kinase. In addition, NGF decreased colchicine toxicity on TH(+) neurons in a manner dependent on MEK but not phosphatidylinositol 3-kinase. The protective effect of NGF against rotenone toxicity was occluded by the microtubule-stabilizing drug taxol. In a MEK-dependent manner, NGF significantly attenuated rotenone- or colchicine-induced microtubule depolymerization and ensuing accumulation of vesicles in the soma and elevation in protein carbonyls. Moreover, other neurotrophic factors such as brain-derived neurotrophic factor and glia cell line-derived neurotrophic factor also reduced rotenone- or colchicine-induced microtubule depolymerization and death of TH(+) through a MEK-dependent mechanism. Thus, our results suggest that neurotrophic factors activate the microtubule-associated protein kinase pathway to stabilize microtubules, and this action significantly attenuates rotenone toxicity on dopaminergic neurons.

During my thesis work I studied the expression and potential function of brain expressed microRNAs (miRNAs) in human prefrontal cortex (PFC). Initially, I used combinatorial computational analysis and microarray data to identify miRNAs that are predicted with high probability to target the human Brain Derived Neurotrophic Factor (BDNF) 3’ Untranslated Region (3’UTR) and are expressed in moderate to high levels in adult human prefrontal cortex. A subset of 10 miRNAs segregating into 5 different miRNA families (miR-30a-d, miR-103/107, miR-16/195, miR-191 and miR-495) met the above criteria. I then designed a protocol to detect these miRNAs with Locked Nucleic Acid (LNA) in situ hybridization in human prefrontal cortex and determine their layer and cellular expression patterns. LNA in situ revealed differential lamina and cellular enrichment of BDNF-related miRNAs. As an example, miR-30a-5p was found to be enriched in large pyramidal neurons of layer 3, which was verified using laser capture microdissection of layer 3 pyramidal neurons and quantitative Real Time Polymerase Chain Reaction (qRT-PCR) following dissection of upper and deeper layers of human PFC. Parallel to this, I used miRNA qRT-PCR to determine the developmental expression of miRNAs using postmortem PFC tissues ranging from embryonic age to old adulthood and compared miRNA to BDNF protein levels. My results revealed a robust inverse correlation between BDNF-related miRNAs and BDNF protein during late maturation and aging of human prefrontal cortex. In vitro luciferase assays and/or lentivirus mediated neuronal miRNA overexpression experiments validated that at least two miRNAs, miR-30a-5p and miR-195, target human BDNF 3’UTR and mediate its translational repression. In the second part of my thesis work I measured levels of miR-30a and miR-195 in the prefrontal cortex of patients with schizophrenia and compared them with levels of BDNF protein and BDNF-related GABAergic mRNAs. According to my results differences in miR-195 levels in a subset of subjects diagnosed with schizophrenia were found to be associated with disease related changes in BDNF protein levels and deficits in BDNF dependent GABAergic gene expression. In the last part of my work I focused on miR-30b, another member of the miR-30 family, which I found to be reduced in the prefrontal cortex of female but not male subjects with schizophrenia. More importantly, disease related changes in miR-30b levels were strongly associated with the age of onset of the disease. Additional experiments in mouse cortex and hippocampus revealed a gender dimorphic expression pattern of this miRNA with higher expression in female brain. Collectively, my results suggest that miRNAs could participate in novel molecular pathways that play an important role during cortical development and maturation and are potentially linked to the pathophysiology of neuropsychiatric disease.

Ultrasound-responsive neurotrophic factor-loaded microbubble- liposome complex: Preclinical investigation for Parkinson's disease treatment

The effect of aerobic exercise at different intensities on Alzheimer's disease (AD) still remains unclear. We investigated the effect of aerobic exercise at different intensities on cognitive and motor functions and neurotrophic factor expression. Thirty-two AD-induced rats were randomly assigned to control (CG), low-intensity (Group I), medium-intensity (Group II), and high-intensity (Group III) exercise groups. Each group, except for the CG, performed aerobic exercise for 20 min a day five times a week. After performing aerobic exercise for 4 weeks, their cognitive and motor functions and neurotrophic factor expression patterns were analyzed and compared between the groups. All variables of cognitive and motor functions and neurotrophic factor expression were significantly improved in Groups I, II, and III compared to those in the CG (p < 0.05). Among the neurotrophic factors, brain-derived neurotrophic factor (BDNF) expression was significantly improved in Group III compared to that in Groups I and II (p < 0.05). In the intra-group comparison of cognitive and motor functions, no significant difference was observed in CG, but the aerobic exercise groups showed improvements. Only Group III showed a significant improvement in the time it took to find eight food items accurately (p < 0.05). Aerobic exercise improved the cognitive and motor functions and neurotrophic factor expression patterns in the AD-induced rat model, with high-intensity aerobic exercise having greater effects on cognitive function and BDNF expression.

PurposeThe skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model.Materials and MethodsSeventy two Sprague-Dawley rats weighing 200–250 gm were enrolled in this study. Muscle denervation model was conducted by transverse resection of a sciatic nerve with the proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of the sciatic nerve followed by four stitches reconnection. These animals were allocated to three groups: control, electrical muscle stimulation, and AFS groups.ResultsNT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology.ConclusionIntramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve regeneration in a long term nerve anastomosis model.

The major aim of this thesis was to assess the role of the neurotrophin BDNF and the imemdiate early gene Arc in the mechanism of action of cocaine. To pursue this purpose we exposed the animals to various drug paradigms. We first injected the animals with different doses of cocaine and sacrificed the animals at different time points. These experiments revealed that acute injection of cocaine produced a finely tuned, time-dependent and regional-selective expression profile of both BDNF and Arc suggesting that they indie partecipate in the rapid action of the psychostimulant. The analysis of repente exposure of the animals to cocaine revealed that precursor (pro-) and mBDNF protein forms were markedly reduced 2 h and 72 h post-injection in the prefrontal cortex. Interestingly, in the striatum we found that repeated cocaine injection increased pro-BDNF levels without altering the mature form of the neurotrophin, thereby suggesting that cocaine differently affects BDNF transcription and translation in a region-selective manner, but might also alter neurotrophin processing. These data further support the notion that the corticostriatal network is highly vulnerable to the effects of cocaine, and suggest that abnormal regulation of BDNF expression could contribute, at least in part, to the functional defects observed in drug abusers. We then decided to evaluate whether repente stress, a major risk factor for cociane relapse, might alter the pattern of BDNF and Arc expression following acute administration of cocaine. we provide evidence that repeated stress prevents cocaine-induced activation of BDNF expression and signaling in rat prefrontal cortex. A single injection of cocaine up-regulates BDNF expression in sham (i.e. unstressed) rats but not in repeatedly stressed rats. Similarly, the expression as well as trafficking of the high affinity BDNF receptor trkB promoted by the psychostimulant is impaired in chronically-stressed rats challenged with cocaine. Moreover, among the different intracellular signaling pathways that can be activated by the neurotrophin, i.e. ERK1/2-, Akt- and PLCγ-pathway, we found that cocaine is able to selectively activate the ERK1/2 pathway in sham animals, but not in rats exposed to repeated stress. These data demonstrate that stress globally interferes with BDNF-mediated signaling responses to cocaine challenge, providing key insights into the molecular basis of stress-cocaine interaction and indicating the critical role of the prefrontal cortex in mediating such interaction. To follow up our studies we decided to employ a new administration paradimg of cocaine in order to separate the direct pharmacological effects of cocaine from those associated with active drug selfadministration. To this end, we employed a yoked control-operant paradigm and sacrificed the animals after a single intravenous (i.v.) cocaine self-administration session. Animals self-administering cocaine (SA, 0.25 mg/0.1 ml saline per infusion, 2-h session) did more active lever-presses than yoked-cocaine (YC) and…

Scientific evidence has demonstrated the power of physical exercise in the prevention and treatment of numerous chronic and/or age-related diseases, such as musculoskeletal, metabolic, and cardiovascular disorders. In addition, regular exercise is known to play a key role in the context of neurodegenerative diseases, as it helps to reduce the risk of their onset and counteracts their progression. However, the underlying molecular mechanisms have not yet been fully elucidated. In this regard, neurotrophins, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glia cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), have been suggested as key mediators of brain health benefits, as they are involved in neurogenesis, neuronal survival, and synaptic plasticity. The production of these neurotrophic factors, known to be increased by physical exercise, is downregulated in neurodegenerative disorders, suggesting their fundamental importance in maintaining brain health. However, the mechanism by which physical exercise promotes the production of neurotrophins remains to be understood, posing limits on their use for the development of potential therapeutic strategies for the treatment of neurodegenerative diseases. In this literature review, we analyzed the most recent evidence regarding the relationship between physical exercise, neurotrophins, and brain health, providing an overview of their involvement in the onset and progression of neurodegeneration.

Objective To investigate the effects of hyperbaric oxygen (HBO) combined with moxibustion on learning and memory functions and the expression of hippocampal neurotrophic factor in rats with Alzheimer′s disease (AD). Methods The AD rat model was established by a single injection of aggregated Aβ25-35 into the hippocampus, and the rats were divided into the model group, the moxibustion group and the HBO combined with moxibustion group. The sham surgical group was injected with the same volume of normal saline at the same site. The learning and memory capacity of the rats was detected by Morris water maze. The positive expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in hippocampus was detected by immunohistochemistry, and the expression of BDNF and NGF proteins in hippocampus was detected by Western blotting. Results Compared with those of the sham surgical group, the escape latency and spatial exploration time of the model group significantly lowered (P<0.01), learning and memory capacity was severely impaired, the expression of BDNF and NGF in the hippocampus significantly decreased (P<0.01), with a neurotrophic deficiency. Following treatment with moxibustion or HBO combined with moxibustion, the learning and memory capacity of the model rats recovered to a certain extent (P<0.05 or P<0.01). The expression of BDNF and NGF in hippocampus was significantly up-regulated (P<0.01), with the therapeutic effects of the HBO combined with moxibustion group being obviously better than those of the moxibustion group (P<0.05). Conclusion HBO combined with moxibustion could significantly improve the learning and memory function of the AD model rats, up-regulate the expression of neurotrophic factor in hippocampus, and the effects were significantly superior to simple moxibustion treatment. Key words: Alzheimer′s disease; Hyperbaric oxygen; Moxibustion; Neurotrophic factor

Growing evidences have revealed that the proforms of several neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3), by binding to p75 neurotrophin receptor and sortilin, could induce neuronal apoptosis and are implicated in the pathogenesis of various neurodegenerative diseases. The glial cell line-derived neurotrophic factor (GDNF), one of the most potent useful neurotrophic factors for the treatment of Parkinson's disease (PD), is firstly synthesized as the proform (proGDNF) like other neurotrophin NGF, BDNF, and NT3. However, little is known about proGDNF expression and secretion under physiological as well as pathological states in vivo or in vitro. In this study, we investigated the expression profile and dynamic changes of proGDNF in brains of aging and PD animal models, with the interesting finding that proGDNF was a predominant form of GDNF with molecular weight of about 36kDa by reducing and nonreducing immunoblots in adult brains and was unregulated in the aging, lipopolysaccharide (LPS), and 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) insult. We further provided direct evidence that accompanied activation of primary astrocytes as well as C6 cell line induced by LPS stimulation, proGDNF was increasingly synthesized and released as the uncleaved form in cell culture. Taken together, our results strongly suggest that proGDNF may be a biologically active protein and has specific effects on the cells close to its secreting site, and a potentially important role of proGDNF signaling in the brains, in the glia-neuronal interaction or in the pathogenesis of PD, should merit further investigation.

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.

Obesity promotes oxidative stress and exacerbates blood-brain barrier disruption after high-intensity exercise

Comparison of the capability of GDNF, BDNF, or both, to protect nigrostriatal neurons in a rat model of Parkinson's disease

Neonatal hypoxia-ischemia (HI) is a risk factor for myelination disturbances, a key factor for cerebral palsy. Cannabidiol (CBD) protects neurons and glial cells after HI insult in newborn animals. We hereby aimed to study CBD’s effects on long-lasting HI-induced myelination deficits in newborn rats. Thus, P7 Wistar rats received s.c. vehicle (HV) or cannabidiol (HC) after HI brain damage (left carotid artery electrocoagulation plus 10% O2 for 112 min). Controls were non-HI pups. At P37, neurobehavioral tests were performed and immunohistochemistry [quantifying mature oligodendrocyte (mOL) populations and myelin basic protein (MBP) density] and electron microscopy (determining axon number, size, and myelin thickness) studies were conducted in cortex (CX) and white matter (WM). Expression of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) were analyzed by western blot at P14. HI reduced mOL or MBP in CX but not in WM. In both CX and WM, axon density and myelin thickness were reduced. MBP impairment correlated with functional deficits. CBD administration resulted in normal function associated with normal mOL and MBP, as well as normal axon density and myelin thickness in all areas. CBD’s effects were not associated with increased BDNF or GDNF expression. In conclusion, HI injury in newborn rats resulted in long-lasting myelination disturbance, associated with functional impairment. CBD treatment preserved function and myelination, likely as a part of a general neuroprotective effect.

In the primary olfactory pathway axons of olfactory neurons (ONs) are accompanied by ensheathing cells (ECs) as the fibres course towards the olfactory bulb. Ensheathing cells are thought to play an important role in promoting and guiding olfactory axons to their appropriate target. In recent years, studies have shown that transplants of ECs into lesions in the central nervous system (CNS) are able to stimulate the growth of axons and in some cases restore functional connections. In an attempt to identify a possible mechanism underlying EC support for olfactory nerve growth and CNS axonal regeneration, this study investigated the production of growth factors and expression of corresponding receptors by these cells. Three techniques immunohistochemistry, enzyme linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess growth factor expression in cultured ECs. Immunohistochemistry showed that ECs expressed nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell-line derived neurotrophic factor (GDNF). ELISA confirmed the intracellular presence of NGF and BDNF and showed that, compared to BDNF, about seven times as much NGF was secreted by ECs. RT-PCR analysis demonstrated expression of mRNA for NGF, BDNF, GDNF and neurturin (NTN). In addition, ECs also expressed the receptors trkB, GFRα-1 and GFRα-2. The results of the experiments show that ECs express a number of growth factors and that BDNF in particular could act both in a paracrine and autocrine manner.

Synergistic effects of NGF, CNTF and GDNF on functional recovery following sciatic nerve injury in rats