Developmental loss of oligodendrocytes exacerbates adult CNS demyelination

Background Multiple sclerosis (MS), a neurodegenerative autoimmune disease characterized by loss of oligodendrocytes and myelin in the brain and spinal cord, results in localized functional deficits. Several risk factors have been associated with MS, however none can fully explain an enhanced susceptibility. Epidemiological data, based on geographical prevalence studies suggest susceptibility is established early in life and frequently long before disease diagnosis implicating developmental events influence adult disease progression. Here we test the hypothesis that loss of mature oligodendrocytes during postnatal development results in enhanced susceptibility to an adult demyelinating insult. Methods A transgenic mouse model was utilized to specifically induce apoptosis in a subset of mature oligodendrocytes (MBP-iCP9) during the first 2 postnatal weeks followed by either a local LPC spinal cord injection or the induction of EAE in the adult. Immunostaining, immunoblotting, behavioral testing, and electron microscopy were utilized to examine the differences between groups. Results We show that during development, oligodendrocyte apoptosis results in transient reductions in myelination and functional deficits that recover after 10-14 days. Compared to animals in which oligodendrocyte development was unperturbed, animals subjected to postnatal oligodendrocyte ablation showed delayed recovery from an LPC lesion. Unexpectedly, the induction and severity of EAE was not significantly altered in animals following oligodendrocyte ablation even though there was a substantial increase in spinal cord tissue damage and CNS inflammation. It is currently unclear why these changes are not reflected in enhanced functional deficits. Conclusions These observations suggest that developmental loss of oligodendrocytes results in long lasting tissue changes that alter its capacity for repair in the adult.


Introduction
Multiple sclerosis (MS) is characterized by oligodendrocyte loss, demyelination, microglial activation, immune cell infiltration, and inflammation that are correlated with functional deficits. A variety of risk factors including gender, genetics, vitamin D deficiency, race and certain viral infections have been associated with MS [1] while epidemiological studies indicate that environmental factors early in life influences the development of the adult disease [2]. The concept that early insults influence the progression of adult disease is not restricted to MS and has been proposed for a number of diseases including Crohn's disease [3], asthma [4], and autism [5], where exposure to an insult such as infectious agents early in development has a lasting influence on the neuroimmune responses [6] and cognitive functions that become apparent following a second adult challenge or insult [7,8]. Mechanistically, this has been proposed to present a priming phenomenon reflecting a phenotypic alteration in the microglial population characterized by increases in interleukin-1 beta (IL-1β) production [9]. It is likely however, that priming also involves other cell types and occurs in response to a broad range of other developmental perturbations.
In the current study, we utilized a transgenic mouse model (MBP-iCP9) which expresses an inducible form of caspase 9 (iCP9) driven by a fragment of the MBP promoter to specifically target apoptosis in a subpopulation of mature oligodendrocytes [10,11] during development. Crosslinking iCP9, through local delivery of a chemical inducer of dimerization (CID), results in activation of the caspase pathway and the apoptosis of mature oligodendrocytes without directly affecting other central nervous system (CNS) cell types [12,13]. Combining early oligodendrocyte ablation with either spinal cord lysophosphatidylcholine (LPC, lysolecithin) lesion or the induction of experimental autoimmune encephalomyelitis (EAE) in adults, we show that while early ablation of 4 oligodendrocytes results in a reduction in myelin and functional deficits, these recover relatively rapidly. Developmental loss of oligodendrocytes does however significantly impair recovery and increases CNS immunoreactivity in mature animals following either LPC or EAE induced damage. Together these studies support the notion that damage to the oligodendrocyte lineages early in development enhances the susceptibility to demyelinating insults in the adult CNS.

Animals and in vivo injections. All the studies comply with the George Washington
University Medical Center Institutional Animal Care and Use Committee guidelines. Both male and female MBP-iCP9 transgenic mice [10] on a C57Black6 background were used throughout this study. Due to the limited number of transgenic pups, we were unable to perform gender specific studies although no obvious differences were detected. For the EAE induction studies, only female mice were used per manufacturer's instruction (Hook Laboratories; # EK-2110).
Pups were injected subcutaneously with 100 mg/Kg body weight of CID (clontech laboratories; #635069) daily for 7 days starting on postnatal day 4 or every other day for a total of 7 injections. CID stock solution was made in 100% ethanol and diluted in equal volume of Polyethylene Glycol (PEG400, Fisher Scientific; #P167) and 1% Tween-20 in PBS. Controls were injected with vehicle lacking CID, where 100% ethanol was added to PEG400 and 1% Tween-20 in PBS. Under terminal sedation, mice were sacrificed 2 weeks after the LPC and 4 weeks after the EAE studies and transcardially perfused with 1xPBS followed by fixative. Animals were fixed with either 4% paraformaldehyde (PFA) for immunefluorescence staining or 4% paraformaldehyde-2% glutaraldehyde-0.1 M sodium Cacodylate (PFA/GA) for Scanning Electron Microscopy (SEM). A minimum of 3 animals were used for each study. 5 LPC surgeries. Male and female pups were injected with vehicle or CID, allowed to mature to 6-7 weeks of age, and a spinal cord LPC lesion performed at T10 and T11 as previously described [11]. Animals were allowed to recover for 14 days before analysis.

EAE induction.
Female vehicle or CID injected pups were allowed to mature to [10][11] weeks at which point EAE was induced accordingly to the manufacturer's guidelines (Hooke Laboratories #EK-2110). Animals showed symptoms of EAE approximately 8 days after induction and were scored on daily basis according to the following criteria: 1 loss of tail tonicity or hind limb weakness to 5 severe paralysis or death (Table 1). Animals scored 3 or higher were given 0.2 ml saline to prevent dehydration.
Open field testing. For functional testing, mice were placed in a Plexiglas open field (Med Associates, St Albans, VT, USA) outfitted with photobeam detectors, and their activity was monitored using the activity monitoring software (Med Associates). Mice were allowed to habituate in the open field for 10 min and total distance traveled and speed were recorded [14].
Tissue processing. Tissues were processed for 3 different procedures. 1) Immunofluorescence staining: animals were fixed with PFA and spinal cords were cryoprotected using 10, 20, and 30% sucrose gradient in 1xPBS solutions; sections were cut at 20 µm. 2) Electron Microscopy: animals were fixed with PFA/GA and spinal cords were cut to 400 µm sections before processing. Sections were then osmicated (1% OsO 4 ) for at least 4 hours followed by 1% uranyl acetate overnight. After gradated dehydration with ethanol and propylene oxide, sections were placed in Epon812 and cut at 1 µm or 120 nm using an ultramicrotome (Leica UC6). Some sections were stained with Solochrome or Toluidine Blue staining to visualize myelin and some were placed on semiconductor grade Si-wafer microtome. 3) Immunoblotting: animals were perfused with cold 1xPBS and spinal cords were flash-frozen in liquid nitrogen and stored at -80 °C.
Immunostaining. Cross sections were rehydrated, blocked, and incubated with primary antibodies overnight followed by appropriate secondary antibodies for an hour prior to mounting. The following primary antibodies were used: MBP (1:300-Abcam; #7349),

Systemic injection of CID ablates oligodendrocytes throughout the CNS
In previous studies local injections of CID into MBP-iCP9 transgenic (TG) mice have been used to specifically ablate a proportion of oligodendrocytes locally in regions of the spinal cord [11], which ultimately recovers, and in the optic nerve [12,13], where recovery is  Figure 2D). No significant regional differences were seen in the response to systemic CID delivery. All regions of the CNS showed reductions in myelin intensity ( Figure 1) and an approximately 50% reduction in the number of CC1+ oligodendrocytes after CID injections (Figures 2A and C).

Systemic oligodendrocytes ablation leads to myelin loss, glial activation and transient functional deficits
To further assess the effect of oligodendrocyte ablation on myelin formation, spinal cord sections from experimental and control animals were stained with Solochrome 3 days after the final injection and the relative intensity assayed. Following CID injections, a reduction of approximately 50% in the level of Solochrome staining was apparent, particularly in the ventral spinal cord ( Figure 3A), suggesting a reduction in myelin formation. Consistent with these observations, ultrastructural analysis showed a 50% reduction in the number of myelinated axons ( Figure 3B) at P21. While vehicle treated animals had on average density of 48/100 µm 2 myelinated axons, in CID treated animals this was reduced to 26/100 µm 2 (p-value 0.0001). In general, the myelin that was present in CID treated animals appeared relatively normal and there were no significant differences in the G ratios. The loss of mature oligodendrocyte affected other glial populations. Three days after CID delivery, astrocytes were activated as indicated by an increase in the GFAP+ processes ( Figure 3C), while microglial morphology changed from ramified to amoeboid and an increase in Iba1 was observed suggesting their activation ( Figure 3D). In addition, the number of PDGFRα+ oligodendrocyte progenitor cells (OPCs) was also increased following ablation of oligodendrocytes ( Figure 3C).
The reduction in oligodendrocytes results in transient functional deficits. Following developmental ablation of mature oligodendrocytes between P4-P18, animals showed 9 reduced mobility at P21 that had largely recovered one week later at P28 (Figure 4). For example, in open field studies, animals subjected to oligodendrocyte ablation showed a significant reduction in the total distance traveled compared to vehicle injected controls at P21 (Figures 4B and C). Similarly, the average speed of movement was significantly reduced in animals following oligodendrocyte ablation compared to vehicle-treated controls ( Figure 4C). The changes in motility were transient and no significant differences between experimental and control animals were detected at P28. These data suggest oligodendrocyte ablation and reduction in CNS myelin impairs motor activity and that the functional recovery is consistent with a normalization of CNS myelin.

Early oligodendrocyte ablation delays adult remyelination.
Previous studies suggested that local ablation of oligodendrocytes resulted in impaired repair following an adult demyelinating lesion [11]. Interpretation of that data was, however complicated due to potential responses to the local CID injections. In the current study, subcutaneous systemic delivery of CID eliminates this complication. The rate of remyelination following a spinal cord LPC lesion was compared in CID and control animals (wild type mice injected with CID and TG mice injected with vehicle) at 6 weeks of age ( Figure 5A). Injection of saline into wild type (WT/-/Saline) or MBP-iCP9 animals showed no significant demyelination ( Figure 5B) as previously reported [11]. Comparison of the lesion area by Solochrome labeling 2 weeks after an LPC lesion demonstrated smaller residual lesions in control MBP-iCP9 animals that were developmentally treated with vehicle compare to those treated with CID ( Figure 5B). To quantify differences in lesion size, the relative proportion of affected dorsal spinal cord white matter was assayed. No differences in total dorsal white matter area were seen between vehicle and CID injected animals ( Figure 5C) and initial lesion formation was similar. By contrast, at 2 weeks post-

Developmental oligodendrocyte ablation increases immune cell activation in EAE
Since developmental ablation of oligodendrocytes impaired remyelination in the LPC model, we examined its effects in the MOG 35-55 -EAE animal model that is both more chronic and more immunologically based. Female MBP-iCP9 animals that had received either vehicle or CID injections during the P4-P18 developmental period were induced with EAE at 11 weeks of age and the development of the disease assayed for 28 days ( Figure   7A). Comparison of the clinical scores showed no significant differences in the timing of onset of disease, the rate of increase in disease severity, or the final level of functional deficits between vehicle and CID treated animals ( Figures 7B and C) suggesting that impairment of remyelination did not exacerbate disease progression. However, histological and immunological analysis of the spinal cord showed significant differences 11 in vehicle and CID treated animals. The lesions in CID treated animals contained more degenerating axons with the residual myelin sheaths that were less well compacted compared to controls ( Figure 7D). The level of inflammation in tissue from CID treated animals was higher than in controls. Elevated levels of astrocyte reactivity were evident through dramatic increases in expression of GFAP throughout the spinal cord (Figure 8).
Similarly, elevated levels of microglial activation were evident by their increased number and the development of ameboid morphology (Figure 9). Together these data suggest The increase in OPCs may reflect a negative feedback loop in which the loss of oligodendrocytes stimulates the production of OPCs. Such a regulatory system has been proposed for OPCs [16,17] and myelin is known to regulate OPC differentiation [18,19]. For instance, studies have shown that exposure to an insult such as an infectious agent early in development may influence neuroimmune responses [6] and cognitive functions in adulthood upon a second challenge or insult [7,8]. This appears to reflect a priming 13 phenomenon in microglia that results in morphological changes and increased microglial interleukin-1 beta (IL-1β) production [9]. Priming may occur as a result of changes in the microenvironment, which may involve other cell types in the CNS and not all the effects of priming require a second insult. Patients that suffer from traumatic brain injury may have  Limp tail and dragging of hind legs. Both hind legs have movement but mouse trips on hind feet. OR no movement in one leg/completely drags one leg, but movement in other leg. OR EAE severity is mild but a strong head tilt that causes the mouse to occasionally fall over. 3 Limp tail and complete paralysis of hind legs. OR one or both hind legs are able to paddle but not able to move forward. OR almost complete paralysis of hind legs. Limp tail and paralysis of one front and one hind leg. OR severe head tilting, walking at the end of the cage, pushing against the cage wall, spinning when pick up at th the tail. 3.5 Limp tail and complete paralysis of hind legs. Mouse moving but when place on its side unable to right itself. Hind together on one side of the body. OR Mouse moving but the hind quarters are flat like a pancake, giving the appearance of a hump in the front qua mouse. 4 Limp tail. Complete hind leg and partial front leg paralysis. Mouse not moving much but appears alert and feedin is recommended. Euthanize if mouse scores 4 for 2 days. With daily SC fluid most C57B may recover to 3.5 or 3.

4.5
Complete hind leg and partial front leg paralysis. No movement. Mouse is not alert and barley responds to contac Euthanasia is recommended. 5 Mouse is spontaneously rolling in the cage. Mouse is dead or euthanized due to severe paralysis.