Michael Namaka

Neurogenesis in postnatal mouse dorsal root ganglia

Neurogenesis continues in various regions of the central nervous system (CNS) throughout life. As the mitogen basic fibroblast growth factor (bFGF) can proliferate neuronal precursors of CNS neurons in culture, and is also upregulated within adult dorsal root ganglia following axotomy, it is possible that the postnatal dorsal root ganglia contain bFGF-responsive neuronal precursors. We undertook cell culture of postnatal mouse dorsal root ganglia to demonstrate neurogenesis. Basic FGF induced a cellular proliferative response in dorsal root ganglia cell culture. After 2 weeks in serum-free medium containing bFGF, neurons were rarely observed. However, following removal of bFGF and addition of trophic factors, many cells were observed that morphologically resembled dorsal root ganglia neurons, stained for neuronal markers, and generated action potentials. Furthermore, bromodeoxyuridine, used as a marker of cytogenesis, was detected in neurofilament-160(+) and/or microtubule-associated protein-2(+) cells that morphologically resembled neurons. In addition to bFGF, epidermal growth factor, nerve growth factor, and sonic hedgehog were also capable of generating spherical cell clusters that contained cells that stained for neuronal markers following the addition of trophic factors. These results suggest that early postnatal dorsal root ganglia contain neural precursors that appear to proliferate in response to various factors and can then be induced to differentiate into neurons. In conclusion, the existence of neural precursors and the possibility of neurogenesis in postnatal dorsal root ganglia may provide a greater range of plasticity available to somatosensory systems during growth or following injury, perhaps to replace ineffectual or dying neurons.

Staphylococcus aureus harbouring Enterotoxin A as a possible risk factor for multiple sclerosis exacerbations

Background: Staphylococcus aureus may produce superantigens that can non-specifically activate CD4+ cells to potentially target the myelin basic protein. Objective: This study examined the association between individuals with multiple sclerosis (MS) and colonization with S. aureus harbouring superantigens. Methods: Nasal swabs were collected from non-MS subjects and patients with MS who had not experienced a relapse in the past six months (MS stable group) and who had suffered a relapse within 30 days of study recruitment (MS exacerbation group). S. aureus was isolated from the anterior nares of participants following standard procedures and staphylococcal superantigen genes (sea, seb, and tsst-1) were detected using standard laboratory PCR techniques. Results: The study enrolled 204 patients, 80 in the non-MS and MS stable groups and 44 patients in the MS exacerbation group. Overall, 27.0% of patients were colonized with S. aureus with no significant differences identified between study groups. Amongst individuals colonized with S. aureus, the prevalence of sea was significantly greater in the MS exacerbation versus non-MS study group (p < 0.05; odds ratio 7.9; 95% confidence interval 1.2–49.5). Conclusions: The ability to rapidly screen patients for the presence of S. aureus producing sea may serve as a useful marker of a potential MS exacerbation.

Transcriptional Regulation of Brain-Derived Neurotrophic Factor (BDNF) by Methyl CpG Binding Protein 2 (MeCP2): a Novel Mechanism for Re-Myelination and/or Myelin Repair Involved in the Treatment of Multiple Sclerosis (MS)

Multiple sclerosis (MS) is a chronic progressive, neurological disease characterized by the targeted immune system-mediated destruction of central nervous system (CNS) myelin. Autoreactive CD4+ T helper cells have a key role in orchestrating MS-induced myelin damage. Once activated, circulating Th1-cells secrete a variety of inflammatory cytokines that foster the breakdown of blood–brain barrier (BBB) eventually infiltrating into the CNS. Inside the CNS, they become reactivated upon exposure to the myelin structural proteins and continue to produce inflammatory cytokines such as tumor necrosis factor α (TNFα) that leads to direct activation of antibodies and macrophages that are involved in the phagocytosis of myelin. Proliferating oligodendrocyte precursors (OPs) migrating to the lesion sites are capable of acute remyelination but unable to completely repair or restore the immune system-mediated myelin damage. This results in various permanent clinical neurological disabilities such as cognitive dysfunction, fatigue, bowel/bladder abnormalities, and neuropathic pain. At present, there is no cure for MS. Recent remyelination and/or myelin repair strategies have focused on the role of the neurotrophin brain-derived neurotrophic factor (BDNF) and its upstream transcriptional repressor methyl CpG binding protein (MeCP2). Research in the field of epigenetic therapeutics involving histone deacetylase (HDAC) inhibitors and lysine acetyl transferase (KAT) inhibitors is being explored to repress the detrimental effects of MeCP2. This review will address the role of MeCP2 and BDNF in remyelination and/or myelin repair and the potential of HDAC and KAT inhibitors as novel therapeutic interventions for MS.

Exploring the role of nerve growth factor in multiple sclerosis: implications in myelin repair.

Multiple sclerosis (MS) is a chronic disease resulting from targeted destruction of central nervous system (CNS) myelin. MS is suggested to be an autoimmune disease involving the pathogenic activation of CD4(+) T cells by a foreign antigen in the peripheral blood. The activated CD4(+) T cells liberate inflammatory cytokines that facilitate the breakdown of the blood-brain barrier (BBB) promoting their passage into the CNS. Inside the CNS, CD4(+) T cells become re-activated by myelin proteins sharing a similar structure to the foreign antigen that initially triggered the immune response. The CD4(+) T cells continue to liberate inflammatory cytokines, such as tumor necrosis factor α (TNFα), which activates macrophages and antibodies responsible for the phagocytosis of myelin. Acute CNS lesions can be re-myelinated, however, the repair of chronic demyelinating lesions is limited, leading to permanent neurological deficits. Although current MS treatments reduce severity and slow disease progression, they do not directly repair damaged myelin. Henceforth, recent treatment strategies have focused on neurotrophins, such as nerve growth factor (NGF) for myelin repair. NGF promotes axonal regeneration, survival, protection and differentiation of oligodendrocytes (OGs) and facilitates migration and proliferation of oligodendrocyte precursors (OPs) to the sites of myelin damage. NGF also directly regulates key structural proteins that comprise myelin. Interestingly, NGF also induces the production of brain-derived neurotrophic factor (BDNF), another integral neurotrophin involved in myelination. The intricate signaling between neurotrophins and cytokines that governs myelin repair supports the role of NGF as a leading therapeutic candidate in white matter disorders, such as MS.

Fatigue and Cognition in Patients with Relapsing Multiple Sclerosis Treated with Interferon Beta

ABSTRACT Introduction: Fatigue and cognitive deficits are common symptoms affecting patients with multiple sclerosis. Methods: The effects of interferon beta on fatigue and cognitive deficits were assessed in 50 patients with relapsing multiple sclerosis (recruited at a single center). The pre-treatment assessments were performed on visits 1 and 2 (Months 0 and 3). Patients started treatment with subcutaneous interferon beta-1a or beta-1b, or intramuscular interferon beta-1a at Month 3, with reassessment at visits 3 and 4 (6 and 12 months, respectively). Co-primary endpoints were change in fatigue (Modified Fatigue Impact Scale) and change in cognition (Brief Repeatable Battery of Neuropsychological Tests) from pre-treatment to visits 3 and 4. Follow-up data were obtained for 40 patients. Results: The pre-treatment demographic and disease characteristics did not differ between groups. Improvements in fatigue levels were reported for patients receiving subcutaneous interferon beta-1a versus patients in the intramuscular interferon beta-1a group (p = .04) and in the interferon beta-1b group (p = .09). Improvements were also reported in five out of 17 cognitive indices for all the treatment groups. Conclusion: The data suggest that interferon beta may reduce fatigue and cognitive deficits in patients with relapsing multiple sclerosis. Larger, randomized, and controlled studies are required to confirm our findings.

Axotomy-induced up-regulation of tumor necrosis factor-alpha in the dorsal root ganglia.

Abstract Objectives: Neuropathic pain is a chronic pain syndrome associated with drug, injury or disease-induced damage or destruction of sensory afferent fibers of the dorsal root ganglia (DRG). Although the exact underlying pathologic mechanisms are not known, pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) are recognized as potential modulators of peripheral and central nervous system inflammatory responses. They play a crucial role in injury and the pathologic development of chronic pain syndromes such as neuropathic pain. Methods: Twenty-four rats were divided into a naive control (n=6), sham (surgery exposing sciatic nerve, n=6), and peripheral nerve lesion group (unilateral axotomy of sciatic nerve, n=12). Results: The results of this study demonstrate a transient up-regulation of TNF-α expression within ipsi- and contralateral DRG following complete unilateral sciatic nerve axotomy as confirmed by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. Elevated expression of TNF-α was noted to occur within the first 7 days post-axotomy, which subsequently normalized to baseline levels by day 14. This transient up-regulation was also associated with a switch in cellular source from predominant satellite cell expression at baseline to that involving satellite cells and abundant numbers of sensory neurons. Discussion: These results support the role of TNF-α in the upstream cascade of cellular events involved in the underlying pathogenesis of neuropathic pain. Strategies targeting the early attenuation of TNF-α within the DRG during the first week post-injury may have significant clinical impact in preventing the downstream cascade of events involved in the underlying cellular pathology of neuropathic pain.

Differential effects of growth factors on oligodendrocyte progenitor migration

Oligodendrocytes are myelinating cells of the CNS that originate as progenitor cells (OP) in discrete areas of the developing brain. During brain development, OP migrate significant distances prior to proliferating and myelinating the axons of the putative white matter tracts. Growth factors play a major regulatory role in the behavior of OP. Specifically, platelet-derived growth factor A (PDGF-A) and fibroblast growth factor 2 (FGF2) are two of the most well characterized regulators of OP development. Both growth factors interact with tyrosine kinase receptors, activating various intracellular signaling pathways. The current study advances our earlier research by comparing the effects of both PDGF-A and FGF2 on OP migration. Our results show that activation of ERK is required for OP migration. These findings correlate well with our previous demonstration of the ERK pathway mediating PDGF-A induced OP migration. We also demonstrate the significance of threshold levels of growth factors and temporal regulation for OP migration. In addition, ERK activation alone is not sufficient to induce OP migration. The current research supports the involvement of the non-ERK mediated signaling pathway in OP migration.

A novel transcriptional regulator of myelin gene expression: implications for neurodevelopmental disorders

Myelination is critical for normal functioning of mammalian central nervous system. Central nervous system myelin is created and maintained by oligodendrocytes. Protein expression patterns change as the oligodendrocyte progenitors differentiate into myelinating oligodendrocytes. Several proteins, including the cell surface proteoglycan NG2, proteolipid protein, myelin basic protein, and myelin-associated glycoprotein are critical for normal myelination. The molecular regulation of myelination is for the most part unknown, although several transcription factors have been identified as regulating myelin protein expression. We have identified a known transcriptional regulator, methyl-CpG-binding protein 2, as regulating myelin specific gene expression in a transgenic mouse. Our findings show a potential role for myelin in the pathophysiology of methyl-CpG-binding protein 2 mutation-associated disorders.

Prenatal inflammation-induced NF-κB dyshomeostasis contributes to renin-angiotensin system over-activity resulting in prenatally programmed hypertension in offspring

Studies involving the use of prenatally programmed hypertension have been shown to potentially contribute to prevention of essential hypertension (EH). Our previous research has demonstrated that prenatal inflammatory stimulation leads to offspring’s aortic dysfunction and hypertension in pregnant Sprague-Dawley rats challenged with lipopolysaccharide (LPS). The present study found that prenatal LPS exposure led to NF-κB dyshomeostasis from fetus to adult, which was characterized by PI3K-Akt activation mediated degradation of IκBα protein and impaired NF-κB self-negative feedback loop mediated less newly synthesis of IκBα mRNA in thoracic aortas (gestational day 20, postnatal week 7 and 16). Prenatal or postnatal exposure of the IκBα degradation inhibitor, pyrollidine dithiocarbamate, effectively blocked NF-κB activation, endothelium dysfunction, and renin-angiotensin system (RAS) over-activity in thoracic aortas, resulting in reduced blood pressure in offspring that received prenatal exposure to LPS. Surprisingly, NF-κB dyshomeostasis and RAS over-activity were only found in thoracic aortas but not in superior mesenteric arteries. Collectively, our data demonstrate that the early life NF-κB dyshomeostasis induced by prenatal inflammatory exposure plays an essential role in the development of EH through triggering RAS over-activity. We conclude that early life NF-κB dyshomeostasis is a key predictor of EH, and thus, NF-κB inhibition represents an effective interventional strategy for EH prevention.

A novel decalcification method for adult rodent bone for histological analysis of peripheral-central nervous system connections.

Histological analysis of bone encased tissue is severely hampered by technical difficulties associated with sectioning calcified tissue. Cryosectioning of bone is possible but requires significant technical adaptation and expensive materials and is often time-consuming. Some decalcifying reagents in common use result in successful cryosectioning in less time but the integrity of the soft tissue of the spinal column is often compromised during processing. This can result in significant loss of cellular detail. In order to find a method that would allow cryosectioning of the bone without loss of structural integrity of the underlying soft tissue we assessed the efficacy of four different decalcifying reagents with respect to their effects on the cellular structure of the myelin of the grey and white matter of the spinal cord. The antigenic integrity of the spinal cord white matter was evaluated using tissue structural integrity and quality of myelin basic protein immunostaining. The result of this research shows that 6% TCA not only decalcifies intact spinal column suitably for cryosectioning but does so without compromising the antigenic integrity of the tissue. The ease of application, speed of processing and a favorable cost-effective profile were secondary benefits noted with the use of the 6% TCA decalcifying solution. The ability to utilize a decalcifying solution that allows for both histomorphometry and immunohistochemistry in the same spinal column segment represents a novel technique that will provide new insights into pathophysiological aspects and therapeutic approaches ispinal cord damage or disease.