Clinically relevant intronic splicing enhancer mutation in myelin proteolipid protein leads to progressive microglia and astrocyte activation in white and gray matter regions of the brain

Microglia are distinguished from other glial cells, such as astrocytes and oligodendrocytes, by the expression of macrophage-associated markers, such as CD11b. Through sampling of the microenvironment, microglia are able to rapidly respond to a disturbance in tissue homeostasis by moving to the potential threat, often in numbers [1, 2]. In most diseases of the central nervous system (CNS), an increased expression of macrophage-associated markers, such as major histocompatibility complex (MHC) class II, has been associated with an activation of microglia. Activated microglia in CNS diseases can damage neurons through the production of neurotoxic substances such as tumor necrosis factor-α (TNF-α) [3]. However, the presence of activated microglia in a CNS disease can be a consequence of a tissue disturbance, such as dying cells (neurons or oligodendrocytes), and not a cause of the cell death.

Beyond the responsive, immunological effector like functions, it is now being increasingly recognized that microglia have a number of important physiological functions in the healthy CNS. The housekeeping functions of microglia in the healthy CNS include: 1) phagocytosis of dying cells and cellular debris (such as myelin); 2) synaptic interactions and synaptic pruning; 3) regulation of neuronal activity; 4) suppression of inflammation mediated by inflammatory monocytes; 5) modulating neurogenesis and oligodendrogenesis (for review see: [4‐8]). Moreover, genetic mutations affecting microglia function are linked to neurological disease (for review see: [9]); including, both neurodevelopmental disorders (MECP2 in the case of Rett syndrome [10]), and neurodegenerative disorders (CD33 and TREM2 in the case of Alzheimer’s disease [11]) for example.

While still poorly understood, it is believed a heterogeneity exists in microglia depending on the region of the CNS where they are found [8]. The most obvious example would be differences in microglia in white matter versus gray matter regions of the brain. In gray matter, microglia have been shown to be involved in synaptic reorganization [12]. In white matter, microglia facilitate the turnover of myelin [13]. There may also be regional differences in the reactive responses of microglia that make some neuron and glia populations particularly vulnerable to neuroinflammatory responses [14]. Therefore, it is important to understand regional heterogeneity in the microenvironment and in the microglia populations that may influence the reactive glia response and pathology that could result from that response.

Focusing on regional heterogeneity of white matter, recent work has shown that alterations in myelin proteins including proteolipid protein (PLP) and 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP) alone can induce a robust microglia response [15‐29]. PLP is the most abundant protein of myelin in the CNS. PLP1 gene encodes for two proteins: PLP, and a smaller splice isoform DM20 that originates from alternative splicing of exon 3B [30]. In the CNS, oligodendrocytes express PLP and DM20 and regulate the expression of these two integral membrane myelin proteins in a developmental fashion. Despite the abundance of PLP in myelin, and the obvious role in neurological disease, PLP1 knockout (KO) mice have only subtle changes in myelin integrity [22, 31, 32]. However, the PLP1 KO mice do develop a progressive axonal pathology [22].

In humans, mutations in the PLP1 gene cause a spectrum of neurological diseases that vary in severity from the milder spastic paraplegia (SPG2) to the severe connatal Pelizaeus-Merzbacher disease (PMD) [33]. Clinically, deletion of an intronic enhancer (ISEdel) within intron 3 of the PLP1 gene is associated with a mild form of PMD that presents with progressive neurological disability [34]. Recently, a knockin (KI) mouse, PLP-ISEdel, was generated to experimentally define the function of this mutation [35]. The mutation was found to reverse the ratio of PLP to DM20 (less PLP and more DM20), with no change in the total amount of protein from the PLP1 gene. Importantly, the ISEdel mutation in PLP leads to myelin abnormalities that become progressively worse with age [35].

The goal of this study was to determine if the PLP-ISEdel mouse model of a clinically relevant mild form of PMD, that neither lacks nor overexpresses PLP, would be sufficient to induce a reactive microglia response, as seen in more aggressive models of PMD [16, 23, 24]. In addition, we sought to determine if there was any regional heterogeneity in the microglia response. To this end, the temporal and spatial profile of the reactive glia response (microglia and astrocyte) was measured in the PLP-ISEdel mouse. The reactive glia response was compared to behavioral changes and axonal injury seen in the PLP-ISEdel mouse. We found an early increase in the reactive glia response in the white matter that progressed to involve gray matter regions of the brain. Our results, together with other clinical and preclinical models of PMD, suggest that clinically relevant mutations in PLP1 are associated with a regionally progressive reactive glia response.

PLP is the major myelin protein of the CNS. Point mutations and deletions/duplications of the PLP1 gene are associated with X-linked dysmyelinating leukodystrophies, PMD and SPG2 [33]. Duplication of the normal PLP1 gene is associated with approximately 70% of PMD cases [44, 45]. Point mutations in the PLP1 gene are a minority of the PMD and SPG2 cases, but can cause the entire spectrum of the PMD and SPG2 clinical manifestations [46]. Recently, we have identified in a group of PMD patients that a deletion in the PLP1 intron 3 splicing enhancer caused a mild form of PMD [34]. The PLP-ISEdel mouse was generated to investigate the mechanism by which loss of the intron 3 splicing enhancer leads to the progressive neurological disability, demyelination, and axonal loss in the PMD patients with this PLP1 gene mutation [35]. As we previously reported, by electron microscopy analysis of the optic nerve, myelin in the PLP-ISEdel mouse forms normally although there are redundant loops of myelin at one month. At three and six months, myelin compaction is progressively abnormal suggesting myelin instability; however, changes in the levels of myelin basic protein and CNP in the optic nerve were not found [35]. We report here the temporal and spatial profile of microglia and astrocyte activation in the PLP-ISEdel mouse and the regional heterogeneity of the glial activation response. We also compared the temporal onset of the glia activation to behavioral changes and to a marker of axonal injury (APP). We found that motor behavior abnormalities were present at the earliest age investigated (two-months-old). In addition we found a reactive glia response at 2-months-old in the PLP-ISEdel mice. While the motor impairments did not worsen in the 4-month-old group of PLP-ISEdel mice, we did find a widespread increase in markers of a reactive glia response with age. In addition, in the 4-month-old PLP-ISEdel mice, a marker of axonal injury was increased.

The motor deficits in the open field test and the impairment in nesting behavior occurred in the PLP-ISEdel mice at two months of age. This is in agreement with our previous report, which found motor impairments using the accelerating rotarod at three months of age in the PLP-ISEdel mice [35]. The motor impairments in the PLP-ISEdel mice were not progressive between two months of age and four months of age, supporting our previous work using the rotarod [35]; however, increasing motor impairments at later stages cannot be ruled out. This is in contrast to progressive motor impairment seen in the PLP-null mouse; however, these motor impairments are not evident until after the mice are 16 months of age [22]. In comparison, the recently generated PLP1dup mice, which have a single duplication of the PLP1 gene as found in the majority of PMD patients, have a progressive motor impairment beginning at approximately four months of age [21]. We found a progressive, age-related change in the search strategy used to explore the open field, as the 4-month-old PLP-ISEdel mice demonstrated increased use of a thigmotaxis behavior. While the thigmotaxis behavior did not appear to reflect anxiety or visual deficits, the possibility that visual deficits influenced the thigmotaxis behavior should not be excluded.

A major finding in this study is the widespread early and progressive astrogliosis in white and gray matter regions of the PLP-ISEdel mice. In the 2-month-old mice, the astrogliosis began in the cerebellum, brain stem and thalamus. By 4-months-old, the PLP-ISEdel mice showed increased astrogliosis throughout the brain. Neuropathological assessments of PMD patients with PLP1 duplication, deletion, and mutations are in agreement with our findings. In the reported cases with mutations in PLP1, astrogliosis was localized to the cerebellum. In the patient cases with PLP1 duplication, strong astrogliosis was found in the striatum, thalamus, cerebellum, and cerebral cortex. It should be noted that the cases reported with mutations in PLP1 were significantly younger at time of death (approximately 20-years-old) than the majority of the reported cases with PLP1 gene duplication (approximately 50-years-old). In one younger case with PLP1 gene duplication (37 years), astrogliosis was less pronounced [47]. Therefore, the astrogliosis in the PLP-ISEdel mice appears to follow a clinically relevant disease progression.

Our findings of robust microglia activation in the CNS in both white and gray matter regions of the PLP-ISEdel mice add to the growing body of literature which suggests that disruptions in PLP protein promote microglia activation [15‐24] (see Table 3). In agreement with our findings, a similar pattern of microglia activation to the PLP-ISEdel mice has been reported in PLP1 transgenic mouse (PLP1tg) [16, 19]. Specifically, the number of IBA1⁺ microglia was significantly increased in the cortex and striatum of 1-month-old PLP1tg mice. In the striatum of the PLP1tg mice, microglia processes were found to follow axonal paths, as we report here for the PLP-ISEdel mice. A progressive age-related increase in the number of IBA1⁺ and CD11b⁺ cells was also reported for PLP1tg mice [16, 19], supporting the findings of the current study (Table 3).

Recently, two therapeutic approaches have been used in the PLP1tg mice that have indicated a possible involvement of gliosis and neuroinflammation in the PLP induced sequelae. Intriguing work from the Martini laboratory has provided strong evidence for microglia/macrophages and adaptive immunity (namely CD8⁺ T cells) in pathological changes in the PLP1tg mice [19, 23, 27‐29]. By crossing the PLP1tg mice to a recombination activating gene-1 (RAG-1) mice, lacking mature T- and B-lymphocytes, they were able to reduce the number of CD11b cells and decrease the pathology in the PLP1tg mice [19]. A second approach involved feeding PLP1tg mice a diet enriched with cholesterol. The cholesterol enriched diet was found to rescue motor defects, but did not prevent the hypomyelination. It was suggested that the positive effects of a cholesterol-enriched diet in the PLP1tg mice could be mediated by maintaining health of oligodendrocytes and axons, as well as reducing gliosis and neuroinflammation [56].

Temporal and regional heterogeneity, which may be associated with the severity of the pathophysiology of different mutant PLP mice, has been reported. Interestingly in the PLP1^jp mice [53], which have missense mutation in the PLP1 gene, there is a progressive age-related increase in microglia that was restricted to white matter regions of the brain [16, 17]. In the gray matter of the PLP1^jp mice, microglia density was increased compared to control mice, but there was no change with age [17]. However, the severe phenotype with short lifespan (around P30) in the PLP1^jp may mask more widespread and progressive microglia activation. For example in the PLP1-null mice, which have the mildest phenotype of the PLP mutant mice, microglia activation is found to be increased around one year of age. Interestingly, the increase in microglia activation corresponded with axonal degeneration but occurred before the motor impairment was evident [26]. While the regional distribution of microglia has not yet been reported in the PLP1dup mice, an increase in microglia number was found in regions of axon degeneration [21]. Moreover, microglia in the PLP1dup mice were found to have internalized degenerating axons [21].

Previously, we reported [35] that axonal degeneration was not seen in the optic nerve of the PLP-ISEdel mouse up to six months of age. However, in the current study we find markers of axonal injury (APP spheroids) in a number of brain regions in the CNS of the 4-month-old PLP-ISEdel mice. One potential explanation for the differences in these finding is the low level of microglia and astrocyte activation in the optic nerve. Previous works suggest that microglia are slow to respond to optic nerve injury [57]. However, in the PLPtg mice, CD11b⁺ cells were found to increase in number in the optic nerve at younger ages compared to other brain regions investigated [19]. APP accumulation represents a marker of disrupted anterograde axonal transport. In the PLP null mouse, APP⁺ swellings were identified in the optic nerve and cerebral white matter at approximately 2-months-old and were not found to be progressive [58]. In the current study we did not stain for APP or markers of glia activation in the optic nerve; therefore any comparison between our current study and previous work would be speculation. Interestingly, the regions of the brain with the most robust APP⁺ spheroids (thalamus and striatum) were also regions of the brain with strong microglia response and astrocyte reactivity. However, several mechanisms may contribute to the axonal degeneration. For example, the PLP-ISEdel mouse has impaired transport of sirtuin 2 from the oligodendrocyte cell body to the myelin membrane [59]. The absence of sirtuin 2, a deacetylase, in the myelin sheath may compromise signaling to the axons. In addition to cytokine secretion, activated microglia may contribute to defects of axonal integrity through sodium channel activation and excitoxicity, as has been shown in multiple sclerosis plaques (for review see [60]). The results presented here are descriptive observations; however, our findings do support the notion that regional heterogeneity in the reactive glia response may lead some areas of the CNS to be more susceptible to degeneration.

Neuroinflammation has been suggested to be more damaging to axons than alterations in myelin integrity, including frank demyelination [58]. Our current finding of gliosis, along with other clinical reports of PMD cases [47], and preclinical models of PMD [16, 19, 23, 24, 26‐29] highlight a potentially clinically relevant, oligodendrocyte independent response that may contribute to the disease progression. While more work is clearly necessary, neuroinflammation may represent a viable therapeutic target for PMD.