Impairment of frequency-specific responses associated with altered electrical activity patterns in auditory thalamus following focal and general demyelination.

Multiple sclerosis is characterized by intermingled episodes of de- and remyelination and the occurrence of white- and grey-matter damage. To mimic the randomly distributed pathophysiological brain lesions observed in MS, we assessed the impact of focal white and grey matter demyelination on thalamic function by directing targeted lysolecithin-induced lesions to the capsula interna (CI), the auditory cortex (A1), or the ventral medial geniculate nucleus (vMGN) in mice. Pathophysiological consequences were compared with those of cuprizone treatment at different stages of demyelination and remyelination. Combining single unit recordings and auditory stimulation in freely behaving mice revealed changes in auditory response profile and electrical activity pattern in the thalamus, depending on the region of the initial insult and the state of remyelination. Cuprizone-induced general demyelination significantly diminished vMGN neuronal activity and frequency-specific responses. Targeted lysolecithin-induced lesions directed either to A1 or to vMGN revealed a permanent impairment of frequency-specific responses, an increase in latency of auditory responses and a reduction in occurrence of burst firing in vMGN neurons. These findings indicate that demyelination of grey matter areas in the thalamocortical system permanently affects vMGN frequency specificity and the prevalence of bursting in the auditory thalamus.

Widespread cortical demyelination of both hemispheres can be induced by injection of pro-inflammatory cytokines via an implanted catheter in the cortex of MOG-immunized rats.

Cortical demyelination is a common finding in patients with chronic multiple sclerosis (MS) and contributes to disease progression and overall disability. The exact pathomechanism that leads to cortical lesions is not clear. Research is limited by the fact that standard animal models of multiple sclerosis do not commonly affect the cortex, or if they do in some variants, the cortical demyelination is rather sparse and already remyelinated within a few days. In an attempt to overcome these limitations we implanted a tissue-compatible catheter into the cortex of Dark Agouti rats. After 14days the rats were immunized with 5µg myelin oligodendrocyte glycoprotein (MOG) in incomplete Freund's Adjuvant, which did not cause any clinical signs but animals developed a stable anti-MOG antibody titer. Then the animals received an injection of proinflammatory cytokines through the catheter. This led to a demyelination of cortical and subcortical areas starting from day 1 in a cone-like pattern spreading from the catheter area towards the subarachnoid space. On day 3 cortical demyelination already expanded to the contralateral hemisphere and reached its peak between days 9-15 after cytokine injection with a widespread demyelination of cortical and subcortical areas of both hemispheres. Clinically the animals showed only discrete signs of fatigue and recovered completely after day 15. Even on day 30 we still were able to detect demyelination in subpial and intracortical areas along with areas of partial and complete remyelination. Loss of cortical myelin was accompanied with marked microglia activation. A second injection of cytokines through the catheter on day 30 led to a second demyelination phase with the same symptoms, but again no detectable motor dysfunction. Suffering of the animals appeared minor compared to standard Experimental Autoimmune Encephalomyelitis and therefore, even long-term observation and repeated demyelination phases seem ethically acceptable.

Involvement of MeCP2 in Regulation of Myelin-Related Gene Expression in Cultured Rat Oligodendrocytes.

Methyl CpG binding protein 2 (MeCP2) is a multifunctional protein which binds to methylated CpG, mutation of which cause a neurodevelopmental disorder, Rett syndrome. MeCP2 can function as both transcriptional activator and repressor of target gene. MeCP2 regulate gene expression in both neuron and glial cells in central nervous system (CNS). Oligodendrocytes, the myelinating cells of CNS, are required for normal functioning of neurons and are regulated by several transcription factors during their differentiation. In current study, we focused on the role of MeCP2 as transcription regulator of myelin genes in cultured rat oligodendrocytes. We have observed expression of MeCP2 at all stages of oligodendrocyte development. MeCP2 knockdown in cultured oligodendrocytes by small interference RNA (siRNA) has shown increase in myelin genes (myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and myelin-associated oligodendrocyte basic protein (MOBP)), neurotrophin (brain-derived neurotrophic factor (BDNF)), and transcriptional regulator (YY1) transcripts level, which are involved in regulation of oligodendrocyte differentiation and myelination. Further, we also found that protein levels of MBP, PLP, DM-20, and BDNF also significantly upregulated in MeCP2 knockdown oligodendrocytes. Our study suggests that the MeCP2 acts as a negative regulator of myelin protein expression.

Sex differences in myelin-associated protein levels within and density of projections between the orbital frontal cortex and dorsal striatum of adult rats: implications for inhibitory control.

Impulsive actions and decisions often lead to undesirable outcomes. Lesion and neuroimaging studies have revealed that the orbital frontal cortex (OFC) and dorsal striatum (dSTR) play key roles in inhibitory control. It has been proposed that greater OFC input into the dSTR reflects enhanced top-down cognitive control and less impulsive responding. We previously reported a sex difference in inhibitory control, such that female rats make fewer impulsive errors than do male rats. The goal of the present study was to investigate differences in the OFC and dSTR of young adult male and female rats. In Experiment 1, we measured levels of two myelin-associated proteins, myelin basic protein (MBP) and myelin proteolipid protein (PLP), in the OFC and dSTR. Western blot data revealed that females had significantly higher levels of both MBP and PLP in the OFC but similar levels in the dSTR as compared to males. In Experiment 2, we infused the anterograde tracer, biotinylated dextran amine (BDA), into the OFC and measured the density of BDA in the dSTR. BDA was visualized using histochemistry followed by light microscopy imaging and densitometry analysis. Density of BDA in the dSTR was significantly greater in females as compared to males indicating that the projections from the OFC to dSTR may be greater in females as compared to males. Our results suggest a potential neuroanatomical sex difference that may contribute to the reported differences in inhibitory control levels of male and female rats.

YY1 negatively regulates mouse myelin proteolipid protein (Plp1) gene expression in oligodendroglial cells.

YY1 (Yin and Yang 1) is a multifunctional, ubiquitously expressed, zinc finger protein that can act as a transcriptional activator, repressor, or initiator element binding protein. Previous studies have shown that YY1 modulates the activity of reporter genes driven by the myelin PLP (proteolipid protein) (PLP1/Plp1) promoter. However, it is known that Plp1 intron 1 DNA contains regulatory elements that are required for the dramatic increase in gene activity, coincident with the active myelination period of CNS (central nervous system) development. The intron in mouse contains multiple prospective YY1 target sites including one within a positive regulatory module called the ASE (anti-silencer/enhancer) element. Results presented here demonstrate that YY1 has a negative effect on the activity of a Plp1-lacZ fusion gene [PLP(+)Z] in an immature oligodendroglial cell line (Oli-neu) that is mediated through sequences present in Plp1 intron 1 DNA. Yet YY1 does not bind to its alleged site in the ASE (even though the protein is capable of recognizing a target site in the promoter), indicating that the down-regulation of PLP(+)Z activity by YY1 in Oli-neu cells does not occur through a direct interaction of YY1 with the ASE sequence. Previous studies with Yy1 conditional knockout mice have demonstrated that YY1 is essential for the differentiation of oligodendrocyte progenitors. Nevertheless, the current study suggests that YY1 functions as a repressor (not an activator) of Plp1 gene expression in immature oligodendrocytes. Perhaps YY1 functions to keep the levels of PLP in check in immature cells before vast quantities of the protein are needed in mature myelinating oligodendrocytes.

B-cell depletion attenuates white and gray matter pathology in marmoset experimental autoimmune encephalomyelitis.

This study investigated the effect of CD20-positive B-cell depletion on central nervous system (CNS) white and gray matter pathology in experimental autoimmune encephalomyelitis in common marmosets, a relevant preclinical model of multiple sclerosis. Experimental autoimmune encephalomyelitis was induced in 14 marmosets by immunization with recombinant human myelin oligodendrocyte glycoprotein in complete Freund adjuvant. At 21 days after immunization, B-cell depletion was achieved by weekly intravenous injections of HuMab 7D8, a human-anti-human CD20 antibody that cross-reacts with marmoset CD20. In vivo magnetic resonance imaging showed widespread brain white matter demyelination in control marmosets that was absent in CD20 antibody-treated marmosets. High-contrast postmortem magnetic resonance imaging showed white matter lesions in 4of the 7 antibody-treated marmosets, but these were significantly smaller than those in controls. The same technique revealed gray matter lesions in 5 control marmosets, but none in antibody-treated marmosets. Histologic analysis confirmed that inflammation, demyelination, and axonal damage were substantially reduced in brain, spinal cord, and optic nerves of CD20 antibody-treated marmosets. In conclusion, CD20-postive B-cell depletion by HuMab 7D8 profoundly reduced the development of both white and gray matter lesions in the marmoset CNS. These data underline the central role of B cells in CNS inflammatory-demyelinating disease.

Induction of progressive demyelinating autoimmune encephalomyelitis in common marmoset monkeys using MOG34-56 peptide in incomplete freund adjuvant.

Experimental autoimmune encephalomyelitis in the neotropical primate common marmoset (Callithrix jacchus) is a relevant autoimmune animal model of multiple sclerosis. T cells specific for peptide 34 to 56 of myelin/oligodendrocyte glycoprotein (MOG34-56) have a central pathogenic role in this model. The aim of this study was to assess the requirement for innate immune stimulation for activation of this core pathogenic autoimmune mechanism. Marmoset monkeys were sensitized against synthetic MOG34-56 peptide alone or in combination with the nonencephalitogenic peptide MOG74-96 formulated in incomplete Freund adjuvant, which lacks microbial components. Experimental autoimmune encephalomyelitis development was recorded by monitoring neurological signs, brain magnetic resonance imaging, and longitudinal profiling of cellular and humoral immune parameters. All monkeys developed autoimmune inflammatory/demyelinating central nervous system disease characterized by massive brain and spinal cord demyelinating white matter lesions with activated macrophages and CD3+ T cells. Immune profiling ex vivo demonstrated the activation of mainly CD3+CD4+/8+CD56+ T cells against MOG34-56. Upon ex vivo stimulation, these T cells produced more interleukin 17A compared with TH1 cytokines (e.g. interferon-gamma) and displayed peptide-specific cytolytic activity. These results indicate that the full spectrum of marmoset experimental autoimmune encephalomyelitis can be induced by sensitization against a single MOG peptide in incomplete Freund adjuvant lacking microbial compounds for innate immune activation and by eliciting antigen-specific T-cell cytolytic activity.

Sequence of oligodendrocyte development in the human fetal telencephalon.

Oligodendrocytes (OL), cells that myelinate axons in the CNS, differentiate from early to late oligodendrocyte progenitor cells (OPC) to become mature OL. Unlike the case in the rodent brain, myelin formation starts prenatally in the human brain, but the sequence of OL development and the onset of myelination are not well understood. We studied the human fetal forebrain at midgestation (17-23 gestational weeks, g.w.) using OL lineage-specific antibodies and mRNA probes. Early OPC were present in a gradient from the subventricular zone to the cortical plate. Their close apposition to radial glia fibers suggests a possible role of these fibers in OPC migration. Late OPC reached peak density in the subplate layer, whereas multipolar cells with the morphology of mature OL were restricted to the emerging white matter. At 20 g.w., myelinated axons were observed in the diencephalon, but not in the telencephalon, consistent with caudal-to-rostral progression of myelination. Interestingly, in organotypic slice cultures of the same gestational ages, the subventricular zone contained a considerably greater number of the mature OL cells, suggesting the presence of inhibitory signals in vivo. Overall, in addition to considerable similarities with rodents, important differences in temporal and spatial distribution and regulatory signals for OL differentiation exist in the human brain.

Distinct cellular expressions of creatine synthetic enzyme GAMT and creatine kinases uCK-Mi and CK-B suggest a novel neuron-glial relationship for brain energy homeostasis.

The creatine/phosphocreatine shuttle system, as catalysed reversibly by creatine kinases, is thought to be essential for the storing and buffering of high phosphate-bound energy in tissues with high energy demand. In the present study, we aimed to clarify the cellular system of creatine biosynthesis and its energy metabolism in the mouse brain by immunohistochemistry for creatine biosynthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), ubiquitous mitochondrial creatine kinase (uCK-Mi) and brain-type cytoplasmic creatine kinase (CK-B). GAMT was expressed highly in oligodendrocytes and olfactory ensheathing glia and moderately in astrocytes, whereas GAMT was very low in neurons and microglia. By contrast, uCK-Mi was expressed selectively in neurons and localized in their mitochondria in dendrites, cell bodies, axons and terminals. The distinct and almost complementary distribution of GAMT and uCK-Mi suggests that the creatine in neuronal mitochondria is derived not only from the circulation, but also from local glial cells associated with these neuronal elements. By contrast, CK-B was selective to astrocytes among glial populations, and was exclusive to inhibitory neurons among neuronal populations. Interestingly, these cells with high CK-B immunoreactivity are known to be highly resistant to acute energy loss, such as hypoxia and hypoglycemia. Considering that phosphocreatine generates ATP much faster than the processes of glycolysis and oxidative phosphorylation, the highly regulated cellular expressions of creatine biosynthetic and metabolic enzymes suggest that the creatine/phosphocreatine shuttle system plays a role in brain energy homeostasis through a novel neuron-glial relationship.

Tolerance induction by acylated peptides: suppression of EAE in the mouse with palmitoylated PLP peptides.

Treatment of SJL mice either before or after challenge with palmitoylated PLP139-151 (PAL139-151) completely suppressed or considerably reduced both acute and relapsing stages of EAE induced with PLP139-151. In the presence of Pertussis toxin, treatment with PAL139-151 was less effective, but treatment with a mixture of PAL139-151 and PAL178-191, the palmitoylated PLP epitope to which T cell recognition spreads, resulted in almost complete protection. Proliferation of lymphocytes from treated mice were sharply reduced, and adoptive transfer of lymph node lymphocytes from treated mice to naive recipients resulted in the reduction of the acute phase of EAE and in delayed relapses following challenge. The results suggest that treatment with PAL139-151 leads to both anergy and the generation of regulatory cells.

Diet, lipids and brain development.

Brain development is a sequential anatomical process characterised by specific well-defined stages of growth and maturation. One of the fundamental and necessary events in the normal development of the central nervous system in vertebrates is the formation of a myelin sheath. It is becoming more evident that this process is influenced by dietary lipids. A number of findings have indicated that the administration of a diet deficient in essential fatty acids during development causes hypomyelination in the rat brain. Our studies have shown that lipids can also play a role in accelerating myelinogenesis in the brain of rats whose mothers had been fed, during pregnancy and lactation, a lipid fraction extracted from yeast grown on n-alkanes. Further studies have shown that accelerated myelinogenesis is connected to a precocious appearance of behavioural reflexes. Thus, the use of particular lipids in human nutrition must be carefully screened for possible effects on brain development.