Lara T. Diemel

Cannabinoid-receptor 1 null mice are susceptible to neurofilament damage and caspase 3 activation

Administered cannabinoids have been shown to ameliorate signs of CNS inflammatory disease in a number of animal models, including allergic encephalomyelitis. More recently, neuroprotective actions have been attributed to activation of the cannabinoid 1 receptor in a number of in vitro and in vivo models. One of these, chronic relapsing experimental allergic encephalomyelitis, is considered a robust analog of multiple sclerosis. In this study, spinal cord tissue from cannabinoid receptor 1 knockout mice was analyzed for neurofilament H and myelin basic protein content, as markers of neurons/axons and myelin respectively, during the course of chronic relapsing experimental allergic encephalomyelitis. Dephosphorylation of a neurofilament H epitope, immunoreactive to the SMI32 antibody, was assessed as a marker of axonal damage and levels of the endpoint cell death mediator caspase 3 were evaluated. It was found that both neurofilament and myelin basic protein levels decrease over the course of disease, indicating concomitant neuronal/axonal loss and demyelination. Loss of each marker was more severe in cannabinoid receptor 1 knockout animals. Increased SMI32 reactivity was observed as disease progressed. SMI32 reactivity was significantly increased in knockout animals over wildtype counterparts, an indication of greater axonal dephosphorylation and injury. Active caspase 3 levels were increased in all animals during disease, with knockout animals displaying highest levels, even in knockout animals prior to disease induction. These results indicate that lack of the cannabinoid receptor 1 is associated with increased caspase activation and greater loss and/or compromise of myelin and axonal/neuronal proteins. The increase of caspase 3 in knockout mice prior to disease induction indicates a latent physiological effect of the missing receptor. The data presented further strengthen the hypothesis of neuroprotection elicited via cannabinoid receptor 1 signaling.

Attenuation of proliferation in oligodendrocyte precursor cells by activated microglia

Activated microglia can influence the survival of neural cells through the release of cytotoxic factors. Here, we investigated the interaction between Toll‐like receptor 4 (TLR4)‐activated microglia and oligodendrocytes or their precursor cells (OPC). Primary rat or N9 microglial cells were activated by exposure to TLR4‐specifc lipopolysaccharide (LPS), resulting in mitogen‐activated protein kinase activation, increased CD68 and inducible nitric oxide synthase expression, and release of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin‐6 (IL‐6). Microglial conditioned medium (MGCM) from LPS‐activated microglia attenuated primary OPC proliferation without inducing cell death. The microglial‐induced inhibition of OPC proliferation was reversed by stimulating group III metabotropic glutamate receptors in microglia with the agonist L‐AP4. In contrast to OPC, LPS‐activated MGCM enhanced the survival of mature oligodendrocytes. Further investigation suggested that TNF and IL‐6 released from TLR4‐activated microglia might contribute to the effect of MGCM on OPC proliferation, insofar as TNF depletion of LPS‐activated MGCM reduced the inhibition of OPC proliferation, and direct addition of TNF or IL‐6 attenuated or increased proliferation, respectively. OPC themselves were also found to express proteins involved in TLR4 signalling, including TLR4, MyD88, and MAL. Although LPS stimulation of OPC did not induce proinflammatory cytokine release or affect their survival, it did trigger JNK phosphorylation, suggesting that TLR4 signalling in these cells is active. These findings suggest that OPC survival may be influenced not only by factors released from endotoxin‐activated microglia but also through a direct response to endotoxins. This may have consequences for myelination under conditions in which microglial activation and cerebral infection are both implicated.

Cannabinoids and neuroprotection in CNS inflammatory disease.

The current failure of potent immunosuppressive agents to control progressive disease in multiple sclerosis has moved a focus from immunotherapy towards the need for neuroprotection. There is increasing evidence for cannabinoid-mediated control of symptoms, which is being more supported by the underlying biology. However there is accumulating evidence in vitro and in vivo to support the hypothesis that the cannabinoid system can limit the neurodegenerative possesses that drive progressive disease, and may provide a new avenue for disease control.

Role for TGF-β1, FGF-2 and PDGF-AA in a myelination of CNS aggregate cultures enriched with macrophages

The increase in myelin basic protein (MBP) synthesis observed in brain aggregate cultures supplemented with macrophages is reflected in elevated supernatant protein levels of the key promoters of oligodendrocyte proliferation, fibroblast growth factor‐2 (FGF‐2) and platelet‐derived growth factor‐AA (PDGF‐AA), during the premyelinating phase. Although supernatant levels of transforming growth factor‐β1 (TGF‐β1), the most abundant growth factor produced at the transcriptional and translational levels by phagocytic macrophages, were reduced at this stage, it was the only growth factor for which mRNA expression was increased significantly in macrophage‐enriched cultures. TGF‐β1, which supports oligodendrocyte differentiation, was increased in the supernatant of macrophage‐enriched cultures only after the onset of myelinogenesis. Hence, standard cultures treated with TGF‐β1 during the premyelinating period reproduced effects of macrophage supplementation, inducing an increase in MBP synthesis and in PDGF‐AA and FGF‐2 bioavailability. A similar increase in MBP synthesis in PDGF‐AA treated cultures emphasises its central role in oligodendrocyte progenitor proliferation. In contrast, FGF‐2 blocked MBP synthesis in the cultures. In cultures treated with anti‐TGF‐β1 antibody before or after the first detection of MBP, supernatant levels of TGF‐β1, FGF‐2, and PDGF‐AA were reduced with resultant inhibition of myelination. Paradoxically, supraphysiological TGF‐β1 treatment after the onset of myelination had the same effect on myelin accumulation. These results indicate an enabling and regulatory role for TGF‐β1 in oligodendrocyte development and, as a source of TGF‐β1, macrophages in the inflammatory multiple sclerosis lesion, may have the potential to promote remyelination by modulating the growth factor repertoire in demyelinating disease.

Temporal analysis of growth factor mRNA expression in myelinating rat brain aggregate cultures: Increments in CNTF, FGF‐2, IGF‐I, and PDGF‐AA mRNA are induced by antibody‐mediated demyelination

Myelinogenesis in rat brain aggregate cultures is associated with a pattern of growth factor mRNA expression comparable to that of the developing brain. The rate of increase in platelet‐derived growth factor‐AA (PDGF‐AA) expression was greatest just before the detection of myelin basic protein (MBP) mRNA in the cultures and remained high thereafter, consistent with in vivo observations. Levels of fibroblast growth factor‐2 (FGF‐2) and of ciliary neurotrophic factor (CNTF) mRNA increased continuously over the period of MBP accumulation. High rates of transforming growth factor β1 (TGF‐β1), insulin‐like growth factor‐I (IGF‐I), and neurotrophin‐3 (NT‐3) expression at early time points during the culture gradually decreased over time, indicative of a key regulatory role during oligodendrocyte development. The addition of demyelinative anti‐myelin oligodendrocyte glycoprotein (anti‐MOG) antibody resulted in a significant increase in MBP peptide fragments with a C‐terminus at phenylalanine 89 indicating proteolytic breakdown of MBP after myelin phagocytosis. Immediately after antibody treatment the expression of CNTF mRNA was significantly increased, compared with controls, while that of FGF‐2 and IGF‐I, and of PDGF‐AA peaked during the early and later stages of recovery respectively. Thus, specific growth factors combine to regulate myelination and remyelination in the aggregates; these data have implications for demyelinating disease in which protective growth factor secretion may be central to regeneration. GLIA 30:342–351, 2000.

Remyelination of cytokine‐ or antibody‐demyelinated CNS aggregate cultures is inhibited by macrophage supplementation

Remyelination in CNS aggregate cultures is determined both by macrophage enrichment and the mode of demyelination. Despite the same degree of myelin loss, accumulation of MBP in anti‐MOG antibody‐demyelinated aggregates overtakes that of controls, while recovery is significantly delayed following IFN‐γ‐induced demyelination. In antibody‐treated cultures, remyelination was associated with a significant increase in culture supernatant levels of TGF‐β1, FGF‐2, and PDGF‐AA as well as an induction of TNF‐α immediately following removal of the demyelinating insult. The impaired recovery in IFN‐γ‐treated cultures, denoted by a significant reduction in TGF‐β1, was reversed by treatment with hrTGF‐β1. Macrophage supplementation of the cultures prior to the addition of either demyelinating agent induced a greater degree of myelin loss followed by incomplete remyelination in both cases. This failure to remyelinate was associated in both groups with a several‐fold elevation in TNF‐α and with modest increases in PDGF‐AA and FGF‐2 in the antibody‐treated cultures. In contrast, macrophage supplementation to mature cultures in the absence of any demyelinating treatment resulted in enhanced accumulation of MBP associated with a promyelinative growth factor and TNF‐α profile similar to that in aggregates enriched with macrophages at the outset of the culture period. Hence, effector elements of the adaptive immune response appear to override promyelinogenic in favor of proinflammatory macrophage factors in mature CNS aggregates, counteracting the potential for myelin repair.

Cannabinoid-mediated neuroprotection following interferon-gamma treatment in a three-dimensional mouse brain aggregate cell culture.

Multiple sclerosis is increasingly recognized as a neurodegenerative disease which is triggered by inflammation in the central nervous system (CNS). Demyelination‐associated axonal or neuronal damage is a primary cause of disability and has thus far not been successfully targeted by available drug therapies. The neuroprotective properties of both endogenous and administered cannabinoids have been shown in in vivo and in vitro models of CNS damage following excitotoxic, oxidative, traumatic and ischaemic insults, with a predominantly apoptotic effector mechanism. In this study a foetal mouse telencephalon aggregate cell culture system was developed to compare tissue from cannabinoid receptor 1 knockout mice with wildtype counterparts. Aggregate formation and neurofilament/myelin basic protein accumulation were dependent on the age of foetal dissection and species used. Following treatment with interferon‐γ, levels of myelin basic protein, neurofilament, neuronal dephosphorylation and caspase 3 activation were assessed in telencephalon tissue in vitro. Cytokine treatment resulted in significant loss of the neuronal marker neurofilament‐H in cannabinoid receptor 1 knockout cultures but not in wildtypes, indicating that presence of the cannabinoid receptor 1 gene can be neuroprotective. Caspase 3 activation was higher in cultures from knockout animals, indicating an apoptotic mechanism of cell death. Dephosphorylated neurofilament levels were significantly elevated in knockout mice, lending support to the premise that neurofilament dephosphorylation is a marker for neuronal damage. Taken together, these results indicate that neuroprotection could be elicited through the cannabinoid receptor 1, and point towards a potential therapeutic role for cannabinoid compounds in demyelinating conditions such as multiple sclerosis.

Myelin phagocytosis and remyelination of macrophage‐enriched central nervous system aggregate cultures

An increased level of myelin basic protein (MBP) degradation peptide 80–89, representative of myelin breakdown, is detected in myelinating foetal rat brain aggregate cultures supplemented with peritoneal macrophages at a time coinciding with the onset of myelination. During the period of myelination, the proportion of activated macrophages/microglia in the aggregates decreases, accompanied by a reduction in the content of MBP degradation products. During the recovery period following a demyelinating episode, the rate of MBP synthesis in antibody‐treated standard aggregates was greater than in their medium controls. However, the rate of MBP accumulation was not as efficient in macrophage‐enriched aggregates and was associated with persistently raised MBP peptide levels. Thus, as occurs in multiple sclerosis lesions, attempts at remyelination appear to be counterbalanced by macrophage‐mediated demyelination, with the continued presence of degraded myelin rendering a local environment that is not fully conducive to remyelination. J Neurosci. Res. 66:1173–1178, 2001.

Myelin basic protein isoforms in myelinating and remyelinating rat brain aggregate cultures

Recent evidence suggests that myelin basic protein (MBP) exon‐2‐containing isoforms play a significant role in the onset of myelination because they are more abundant during early development. The pattern of expression of MBP exon‐2‐containing isoforms was studied in rat brain aggregate cultures during myelination to draw comparisons with the developing brain and at remyelination after demyelinative treatment. The pattern of MBP isoform expression in the aggregate cultures was found to be similar to that of the brain and was recapitulated after demyelination with antimyelin antibodies. Macrophage enrichment, resulting in increased accumulation of total MBP in the cultures, did not alter the isoform distribution. Both control and enriched cultures expressed a 16‐kDa protein (26 ± 9.8% of total MBP for control samples) that reacted with MBP antisera at the onset of myelination (day in vitro 14) but was barely detectable by day in vitro 21. The expression of this protein, also present in postnatal day 6 rat brain but no longer by day 11, has been predicted by reverse transcription polymerase chain reaction in embryonic mouse brain. The results of the present study reinforce the value of the aggregate culture system as a versatile yet accurate model of myelination and remyelination. J. Neurosci. Res. 56:241–247, 1999.