Papers by Margaret Fahnestock
Springer eBooks, 2002
Do seizures change the brain? Studies on the kindling model--a widely used animal model of epilep... more Do seizures change the brain? Studies on the kindling model--a widely used animal model of epilepsy--suggest that they do. Dr. Racine, one of the pioneers in the kindling field, describes the basic phenomena of kindling, and discusses the possible roles of cell growth and cell death in this model.
Research Square (Research Square), Aug 27, 2020
Background: Circulating autoantibodies and sex-dependent discrepancy in prevalence are unexplaine... more Background: Circulating autoantibodies and sex-dependent discrepancy in prevalence are unexplained phenomena of Alzheimer's disease (AD). Using the 3xTg-AD mouse model, we reported that adult males show early manifestations of systemic autoimmunity, increased emotional reactivity, enhanced expression of the histone variant macroH2A1 in the cerebral cortex, and loss of plaque/tangle pathology. Conversely, adult females display less severe autoimmunity and retain their AD-like phenotype. This study examines the link between immunity and other traits of the current 3xTg-AD model. Methods: Young 3xTg-AD and wild-type mice drank a sucrose-laced 0.4 mg/ml solution of the immunosuppressant cyclophosphamide on weekends for 5 months. After behavioral phenotyping at 2 and 6 months of age, we assessed organ mass, serologic markers of autoimmunity, molecular markers of early AD pathology and expression of genes associated with neurodegeneration. Results: Chronic immunosuppression prevented hematocrit drop and reduced soluble Aβ in 3xTg-AD males while normalizing the expression of histone variant macroH2A1 in 3xTg-AD females. This treatment also reduced hepatosplenomegaly, lowered autoantibody levels, and increased the effector T cell population while decreasing the proportion of regulatory T cells in both sexes. Exposure to cyclophosphamide, however, neither prevented reduced brain mass and BDNF expression nor normalized increased tau and anxiety-related behaviors. The results suggest that systemic autoimmunity increases soluble Aβ production and affects transcriptional regulation of macroH2A1 in a sex-related manner. Despite the complexity of multisystem interactions, 3xTg-AD mice can be a useful in vivo model for exploring the regulatory role of autoimmunity in the etiology of AD-like neurodegenerative disorders.
Neuroscience, Dec 1, 2002
AbstractöNeurotrophin-3 (NT-3), a member of the neurotrophin family of neurotrophic factors, is i... more AbstractöNeurotrophin-3 (NT-3), a member of the neurotrophin family of neurotrophic factors, is important for cell survival, axonal growth and neuronal plasticity. Epileptiform activation can regulate the expression of neurotrophins, and increases or decreases in neurotrophins can a¡ect both epileptogenesis and seizure-related axonal growth. Interestingly, the expression of nerve growth factor and brain-derived neurotrophic factor is rapidly up-regulated following seizures, while NT-3 mRNA remains unchanged or undergoes a delayed down-regulation, suggesting that NT-3 might have a di¡erent function in epileptogenesis. In the present study, we demonstrate that continuous intraventricular infusion of NT-3 in the absence of kindling triggers mossy ¢ber sprouting in the inner molecular layer of the dentate gyrus and the stratum oriens of the CA3 region. Furthermore, despite this NT-3-related sprouting e¡ect, continuous infusion of NT-3 retards the development of behavioral seizures and inhibits kindling-induced mossy ¢ber sprouting in the inner molecular layer of the dentate gyrus. We also show that prolonged infusion of NT-3 leads to a decrease in kindling-induced Trk phosphorylation and a down-regulation of the high-a⁄nity Trk receptors, TrkA and TrkC, suggesting an involvement of both cholinergic nerve growth factor receptors and hippocampal NT-3 receptors in these e¡ects. Our results demonstrate an important inhibitory role for NT-3 in seizure development and seizure-related synaptic reorganization.
IBRO Reports, Sep 1, 2019
Sesame (Sesamum indicum Linn.) is one of the tropical oil crops mainly found in Thailand, China, ... more Sesame (Sesamum indicum Linn.) is one of the tropical oil crops mainly found in Thailand, China, India, and Africa. It is an important culinary and traditional Chinese medicine. Several lines of research have confirmed its medicinal properties in antimicrobial, anticancer, anti-inflammation, and antioxidant. It contains nutraceutical sources of lignans especially sesamol. The exact mechanism of sesamol involved in neuroprotection still remains to be elucidated. Herein, the neuroprotective effects of sesamol against H 2 O 2 -induced oxidative stress were investigated. The results indicated that pretreatment with 1 M sesamol significantly increased H 2 O 2 -induced cell viability, as determined by MTT and Annexin V apoptosis detection. Protein expression of apoptotic markers (BCL-2 and BAX) was significantly altered in the event of oxidative stress. Sesamol-pretreated cells elicited significant downregulation of BAX apoptotic protein and upregulation of anti-apoptotic BCL-2. Moreover, intracellular reactive oxygen species (ROS) generation induced by H 2 O 2 was determined by DCFDA assay and further confirmed by fluorescence microscope. The ROS production was decreased by pretreatment with 1 M sesamol compared to the H 2 O 2 alone. Meanwhile, the levels of antioxidant markers (SOD and CAT) were significantly increased in sesamol-pretreated cells due to oxidative stress. To better understand the molecular mechanisms of sesamol involved in prevention of neurodegeneration, protein expression levels of SIRT1, SIRT3, and FOXO3a were evaluated. Surprisingly, sesamol clearly maintained SIRT1, SIRT3, and FOXO3a under oxidative stress. These data suggest that sesamol protects neuronal cells against oxidative stress through SIRT1-SIRT3-FOXO3a signaling pathway. Besides, Molecular docking data also revealed that sesamol could be docked well with SIRT1, which is similar to the reference compound. Hence, this bioactive compound might be one of the promising natural agents for the prevention and/or treatment of neurodegenerative diseases.
The FASEB Journal, Apr 1, 2013
Alzheimer's disease is characterized by loss of synapses and neurons in specific areas of the... more Alzheimer's disease is characterized by loss of synapses and neurons in specific areas of the brain, leading to loss of learning, memory and cognitive function. Among the most vulnerable neurons in Alzheimer's disease are basal forebrain cholinergic neurons. Degeneration of cholinergic projections to cortex and hippocampus is responsible for learning and memory deficits. Basal forebrain cholinergic neurons rely on two target‐derived neurotrophic factors, nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF), to maintain survival, differentiation, connectivity and function. NGF and BDNF are dysregulated in Alzheimer's disease by very different mechanisms. NGF is found in the brain in its precursor form, proNGF, which accumulates in Alzheimer's disease and becomes toxic to basal forebrain cholinergic neurons. BDNF, on the other hand, is decreased in Alzheimer's disease at the transcriptional level, causing cholinergic dysfunction and synapse loss. The molecular pathways for BDNF down‐regulation have been partially elucidated, and recent work demonstrates that lifestyle changes can raise BDNF levels and rescue age‐related cognitive impairment. Nevertheless, a full understanding of the causes and mechanisms of basal forebrain cholinergic neuron degeneration in Alzheimer's disease remains a challenge.
Ultrasound in Medicine and Biology, 1989
Mechanisms of action of ultrasound on cell membranes were studied on two murine C1300 neuroblasto... more Mechanisms of action of ultrasound on cell membranes were studied on two murine C1300 neuroblastoma cell lines of minimum genetic diversity. Cavitation was established in rotating polystyrene centrifuge tubes by 1 MHz cw ultrasound; exposure time was 5 minutes. NS20Y and N2A cells exposed in suspension responded similarly by S6Rb+ transport and Na+-K+-ATPase activity assays, but differently by trypan blue dye exclusion and lysis assays. This indicates similar overall damage to the cell membranes, despite use of trypsin to release N2As only. Primary evidence of damage was lysis of NS20Ys and permeabilization of N2As. These results indicate that the same ultrasound exposure conditions can produce different effects in cells that differ in their membrane properties.
Current Topics in Microbiology and Immunology, 1991
Most of our knowledge about NGF comes from extensive study of the mouse submaxillary gland protei... more Most of our knowledge about NGF comes from extensive study of the mouse submaxillary gland protein. NGF from this source is isolated as a high molecular weight complex consisting of beta-NGF and two subunits, alpha and gamma, belonging to the kallikrein family of serine proteases. There are few other tissues where NGF is found in sufficient quantities for protein purification and study, although new molecular biological techniques have accelerated the study of NGFs from a variety of species and tissues. Mouse submaxillary gland NGF is synthesized as a large precursor that is cleaved at both N- and C-terminals to produce mature NGF. This biologically active molecule can be further cleaved by submaxillary gland proteases. The roles of the alpha and gamma subunits in the processing of the beta-NGF precursor, the modulation of the biological activity of beta-NGF, and the protection of mature beta-NGF from degradation have been well studied in the mouse. However, the apparent lack of alpha and gamma subunits in most other tissues and species and the existence of a large family of murine kallikreins, many of which are expressed in the submaxillary gland, challenge the relevance of murine high molecular weight NGF as a proper model for NGF biosynthesis and regulation. It is important therefore to identify and characterize other NGF complexes and to study their subunit interactions, biosynthesis, processing, and regulation. This review points out a number of other species and tissues in which the study of NGF has just begun. At this time, there exist many more questions than answers regarding the presence and the functions of NGF processing and regulatory proteins. By studying NGF in other species and tissues and comparing the processing and regulation of NGF from several sources, we will discover the unifying concepts governing the expression of NGF biological activity.
European Journal of Neuroscience, Jul 1, 2004
Genetic deficits have been discovered in human epilepsy, which lead to alteration of the balance ... more Genetic deficits have been discovered in human epilepsy, which lead to alteration of the balance between excitation and inhibition, and ultimately result in seizures. Rodents show similar genetic determinants of seizure induction. To test whether seizure-prone phenotypes exhibit increased seizure-related morphological changes, we compared two standard rat strains (Long-Evans hooded and Wistar) and two specially bred strains following status epilepticus. The special strains, namely the kindling-prone (FAST) and kindling-resistant (SLOW) strains, were selectively bred based on their amygdala kindling rate. Although the Wistar and Long-Evans hooded strains experienced similar amounts of seizure activity, Wistar rats showed greater mossy fiber sprouting and hilar neuronal loss than Long-Evans hooded rats. The mossy fiber system was affected differently in FAST and SLOW rats. FAST animals showed more mossy fiber granules in the naïve state, but were more resistant to seizure-induced mossy fiber sprouting than SLOW rats. These properties of the FAST strain are consistent with those observed in juvenile animals, further supporting the hypothesis that the FAST strain shares circuit properties similar to those seen in immature animals. Furthermore, the extent of mossy fiber sprouting was not well correlated with sensitivity to status epilepticus, but was positively correlated with the frequency of spontaneous recurrent seizures in the FAST rats only, suggesting a possible role for axonal sprouting in the development of spontaneous seizures in these animals. We conclude that genetic factors clearly affect seizure development and related morphological changes in both standard laboratory strains and the selectively bred seizure-prone and seizure-resistant strains.
Journal of Neurochemistry, May 1, 2008
Several factors have been proposed to account for poor motor recovery after prolonged denervation... more Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, downregulated neuro-trophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.
Free Radical Biology and Medicine, May 1, 2023
PubMed, Dec 1, 2021
Profound and early basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Alzhei... more Profound and early basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Alzheimer’s disease. BFCNs depend for survival and function on retrograde axonal transport of neurotrophins like nerve growth factor (NGF). We have recently shown that the retrograde axonal transport of proNGF, the form of NGF found in the brain, is reduced with age in cultured BFCNs. ProNGF loss coincides with loss of its receptor, tropomyosin‐related kinase A (TrkA), while p75NTR levels remain unchanged. We sought to determine whether mechanisms related to oxidative stress, a longstanding hypothesized contributor to Alzheimer’s disease, account for these reductions.
PubMed, Aug 1, 1994
1. Kallikreins are trypsin-like serine proteases which have been implicated in the biosynthesis o... more 1. Kallikreins are trypsin-like serine proteases which have been implicated in the biosynthesis of a number of growth factors and hormones. Mastomys, an African rodent, is the only mammal other than mouse known to contain high levels of kallikreins and growth factors in its submandibular gland. Mastomys has a large kallikrein family similar to mouse and rat. Cloning and sequencing of several of these cDNAs demonstrates a high degree of homology with mouse and rat kallikreins, but careful analysis suggests that it will not be possible to determine the function of these kallikreins by sequence information alone. 2. It is not known whether a Mastomys kallikrein processes and binds to nerve growth factor (NGF) as in mouse submandibular gland. Mastomys NGF is bound to a protein similar in size, charge, and lack of esterase activity to alpha-NGF, a mouse kallikrein. However, the Mastomys NGF complex does not contain a proteolytically active kallikrein as does the mouse NGF complex. Thus, the identification of kallikreins as growth factor processing enzymes and the presence of a kallikrein family member in high-molecular-weight complexes in species other than mouse is still in question.
Journal of Labelled Compounds and Radiopharmaceuticals, 2002
More than 1000 laboratories worldwide use phosphorimager systems for radioisotopic gel and blot q... more More than 1000 laboratories worldwide use phosphorimager systems for radioisotopic gel and blot quantification. This method is extraordinary sensitive and has a wide dynamic range, and it is therefore often preferred to conventional film-based analysis. We checked the accuracy of our storage phosphor screen by exposing the same 32 P-labelled blot in different positions on the same screen. The first exposure took place at the top of the screen, an area used more often; the second exposure at a relatively little-used area of the screen. As the screens are said to last indefinitely, regardless of how often they are used, we did not expect a difference in the results. In fact, we found a significant impact of the position of the gel on the results. The position that was used more often gave inaccurate results; in contrast, the unused area of the screen gave data that were confirmed by X-ray film autoradiography. We conclude that, contrary to described properties, a deterioration of the phosphor screens takes place with repeated use. To avoid obtaining invalid data, we recommend frequent checks of the screens.
Cells, Aug 4, 2021
Nerve growth factor (NGF) and its precursor form, proNGF, are critical for neuronal survival and ... more Nerve growth factor (NGF) and its precursor form, proNGF, are critical for neuronal survival and cognitive function. In the brain, proNGF is the only detectable form of NGF. Dysregulation of proNGF in the brain is implicated in age-related memory loss and Alzheimer's disease (AD). AD is characterized by early and progressive degeneration of the basal forebrain, an area critical for learning, memory, and attention. Learning and memory deficits in AD are associated with loss of proNGF survival signalling and impaired retrograde transport of proNGF to the basal forebrain. ProNGF transport and signalling may be impaired by the increased reactive oxygen and nitrogen species (ROS/RNS) observed in the aged and AD brain. The current literature suggests that ROS/RNS nitrate proNGF and reduce the expression of the proNGF receptor tropomyosin-related kinase A (TrkA), disrupting its downstream survival signalling. ROS/RNS-induced reductions in TrkA expression reduce cell viability, as proNGF loses its neurotrophic function in the absence of TrkA and instead generates apoptotic signalling via the pan-neurotrophin receptor p75 NTR . ROS/RNS also interfere with kinesin and dynein motor functions, causing transport deficits. ROS/RNS-induced deficits in microtubule motor function and TrkA expression and signalling may contribute to the vulnerability of the basal forebrain in AD. Antioxidant treatments may be beneficial in restoring proNGF signalling and axonal transport and reducing basal forebrain neurodegeneration and related deficits in cognitive function.
Journal of comparative neurology, Jun 4, 2013
Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and mai... more Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and maintenance of distinct populations of neurons. We have generated a line of transgenic mice in which NGF is expressed under the control of the smooth muscle a-actin promoter. These transgenic mice have augmented levels of NGF protein in the descending colon and urinary bladder, so these tissues display increased densities of NGF-sensitive sympathetic efferents and sensory afferents. Here we provide a thorough examination of sympathetic and sensory axonal densities in the descending colon and urinary bladder of NGF transgenic mice with and without the expression of the p75 neurotrophin receptor (p75NTR). In response to elevated NGF levels, sympathetic axons (immunostained for tyrosine hydroxylase) undergo robust collateral sprouting in the descending colon and urinary bladder of adult transgenic mice (i.e., those tissues having smooth muscle cells); this sprouting is not augmented in the absence of p75NTR expression. As for sensory axons (immunostained for calcitonin gene-related peptide) in the urinary bladders of transgenic mice, fibers undergo sprouting that is further increased in the absence of p75NTR expression. Sympathetic axons are also seen invading the sensory ganglia of transgenic mice; these fibers form perineuronal plexi around a subpopulation of sensory somata. Our results reveal that elevated levels of NGF in target tissues stimulate sympathetic and sensory axonal sprouting and that an absence of p75NTR by sensory afferents (but not by sympathetic efferents) leads to a further increase of terminal arborization in certain NGF-rich peripheral tissues.
Molecular Brain Research, Apr 1, 1988
The kallikrein genes and their expression in the salivary glands of mouse, the African rat Mastom... more The kallikrein genes and their expression in the salivary glands of mouse, the African rat Mastomys natalensis and human were compared. The Mastomys kallikrein genes comprise a family of genes similar to those of mouse. Androgen markedly enhances transcription of glandular nerve growth factor (NGF) and kallikrein in both male and female Mastomys suggesting the presence of testosterone regulated kallikrein genes for growth factor precursor-processing in both sexes. In contrast. although a kallikrein transcript was detected in human salivary glands of the same size as the mouse or Mastomys transcript no difference in the amount of transcript was seen in adult male or female. The absence of kallikrein genes regulated by testosterone and of NGF transcripts in the human implies that there is no human equivalent of the mouse salivary 7S NGF complex
Neurobiology of Disease, Dec 1, 2017
Evidence from human neuropathological studies indicates that the levels of the neurotrophins nerv... more Evidence from human neuropathological studies indicates that the levels of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are compromised in Alzheimer's disease. However, the causes and temporal (pathology-dependent) evolution of these alterations are not completely understood. To elucidate these issues, we investigated the McGill-R-Thy1-APP transgenic rat, which exhibits progressive intracellular and extracellular amyloid-beta (Aβ) pathology and ensuing cognitive deficits. Neurochemical analyses revealed a differential dysregulation of NGF and BDNF transcripts and protein expression. While BDNF mRNA levels were significantly reduced at very early stages of amyloid pathology, before plaques appeared, there were no changes in NGF mRNA expression even at advanced stages. Paradoxically, the protein levels of the NGF precursor were increased. These changes in neurotrophin expression are identical to those seen during the progression of Alzheimer's disease. At advanced pathological stages, deficits in the protease cascade controlling the maturation and degradation of NGF were evident in McGill transgenic rats, in line with the paradoxical upregulation of proNGF, as seen in Alzheimer's disease, in the absence of changes in NGF mRNA. The compromise in NGF metabolism and BDNF levels was accompanied by downregulation of cortical cholinergic synapses; strengthening the evidence that neurotrophin dysregulation affects cholinergic synapses and synaptic plasticity. Our findings suggest a differential temporal deregulation of NGF and BDNF neurotrophins, whereby deficits in BDNF mRNA appear at early stages of intraneuronal Aβ pathology, before alterations in NGF metabolism and cholinergic synapse loss manifest.
International Journal of Molecular Sciences, Mar 9, 2017
Nerve growth factor (NGF) promotes the survival and differentiation of neurons. NGF is initially ... more Nerve growth factor (NGF) promotes the survival and differentiation of neurons. NGF is initially synthesized as a precursor, proNGF, which is the predominant form in the central nervous system. NGF and proNGF bind to TrkA/p75NTR to mediate cell survival and to sortilin/p75NTR to promote apoptosis. The ratio of TrkA to p75NTR affects whether proNGF and mature NGF signal cell survival or apoptosis. The purpose of this study was to determine whether the loss of TrkA influences p75NTR or sortilin expression levels, and to establish whether proNGF and mature NGF have a similar ability to switch between cell survival and cell death. We systematically altered TrkA receptor levels by priming cells with NGF, using small interfering RNA, and using the mutagenized PC12nnr5 cell line. We found that both NGF and proNGF can support cell survival in cells expressing TrkA, even in the presence of p75NTR and sortilin. However, when TrkA is reduced, proNGF signals cell death, while NGF exhibits no activity. In the absence of TrkA, proNGF-induced cell death occurs, even when p75NTR and sortilin levels are reduced. These results show that proNGF can switch between neurotrophic and apoptotic activity in response to changes in TrkA receptor levels, whereas mature NGF cannot. These results also support the model that proNGF is neurotrophic under normal circumstances, but that a loss in TrkA in the presence of p75NTR and sortilin, as occurs in neurodegenerative disease or injury, shifts proNGF, but not NGF, signalling from cell survival to cell death.
Journal of comparative neurology, Jul 19, 2011
Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors ... more Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non-neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP-positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury. INDEXING TERMS neurotrophin; nerve growth factor; transgene; brain; peripheral nerve; injury Nerve growth factor (NGF), one of several neurotrophins of the vertebrate nervous system, was first localized in the male mouse submaxillary gland (Levi-Montalcini and Cohen, 1960). Later studies found that sympathetic ganglia, as well as target sites of sympathetic innervation (e.g., irides, heart atria, and prostate), have detectable levels of NGF mRNA and protein (Korsching and Thoenen, 1983a; Heumann et al., 1984; Shelton and Reichardt, 1984). The presence of NGF protein in sympathetic neurons has been linked to the binding and retrograde transport of this neurotrophin from target tissues, rather than to endogenous
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Papers by Margaret Fahnestock