Alicia L. Degano

Immune-mediated mechanisms have emerged as important contributors to neurodevelopmental vulnerability in a subset of autism spectrum disorder (ASD) cases. Circulating IgG antibodies against myelin-associated glycoprotein (MAG) have been reported in individuals with ASD and their mothers; however, the pathogenic relevance of these antibodies and the contribution of MAG signaling to neurodevelopment remain unclear. Here, we investigated whether disruption of MAG function during early postnatal life is sufficient to alter cerebellar development and induce ASD-relevant behavioral alterations. Using complementary genetic and immunological approaches, we show that constitutive deletion of Mag or transient postnatal blockade with a function-blocking anti-MAG antibody induces region-specific alterations in cerebellar development. MAG disruption results in excessive proliferation of granule cell precursors followed by delayed, region-restricted neuronal death, together with persistent abnormalities in Purkinje neuron number and dendritic maturation. These structural changes occur in the absence of major or persistent demyelination, consistent with dysregulation of myelin-associated developmental signaling rather than myelin loss. Importantly, early postnatal passive immunization with anti-MAG IgG is sufficient to induce significant impairments in social communication, sociability, and social recognition, recapitulating core behavioral domains relevant to ASD. Together, these data provide experimental evidence that immune-mediated interference with a myelin-associated signaling molecule can disrupt cerebellar development during critical postnatal windows and produce long-lasting behavioral consequences. Our findings identify MAG as a previously underappreciated regulator of neurodevelopment and support a model in which antibody-mediated perturbation of myelin-derived instructive signaling contributes to ASD-relevant phenotypes, aligning with emerging frameworks of immune-linked neurodevelopmental vulnerability. Highlights: Disruption of myelin-associated glycoprotein (MAG) activity during early postnatal life alters cerebellar development in a region-specific manner. Genetic deletion or antibody-mediated blockade of MAG function induces granule cell dysregulation and Pkn structural abnormalities without overt demyelination. Passive immunization of anti-MAG antibodies is sufficient to induce autism-like behavioral phenotypes, supporting a causal immune-mediated mechanism. These findings identify myelin-associated signaling as a novel contributor to neurodevelopmental dysfunction and align with the maternal autoantibody–related ASD framework.

Rett Syndrome (RTT) is a neurodevelopmental disorder mostly caused by mutations in the X-linked gene, MeCP2, which encodes for methyl-CpG binding protein 2 (MeCP2). MeCP2 is member of a family of methyl binding proteins that control the expression of several genes according to the genomic context. Emerging evidence suggests that immune dysfunctions would actively contribute to the pathogenesis of RTT. Macrophages are key effector cells that participate in several critical aspects of immune responses. The aim of our work was to assess the response of macrophages in vitro in the context of polarizing stimuli. We used bone marrow-derived macrophages (BMDM) obtained from MeCP2 ^308/y mice, a mouse model that carries a truncated form of MeCP2. Since MeCP2 is expressed as a “partially functional” protein in humans with RTT it becomes crucial to establish how the presence of a mutant form of MeCP2 affects immune responses to support the normal homeostasis of individuals. MeCP2 deficiency induced exacerbation of pro-inflammatory mediators and deficient immune regulatory responses under polarizing conditions. These findings suggest that MeCP2 plays a role in the establishment of macrophage polarization in the context of immune activation. Present results may have important implications in understanding RTT pathogenesis and for developing potential treatments. Conflict of interest: The authors declare no conflicts of interest.

Background: Rett syndrome is a severe and progressive neurological disorder linked to mutations in the MeCP2 gene located on the X chromosome. So far it has not been established how the presence of a mutant form of MeCP2 can maintain essential regulation of immune responses to support the normal homeostasis of individuals. Since MeCP2 is mostly expressed as a “partially functional” protein in humans with RTT, the aim of our work was to evaluate whether a mutation in MeCP2 interferes with the induction of neuroinflammatory responses in real time. Methods: We used MeCP2 ^308/y mouse model (MUT) and exposed it to an autoimmune challenge, experimental autoimmune encephalomyelitis (EAE). WT and MUT mice were immunized with CFA-MOG or CFA alone (control) and clinical scores were evaluated daily. Animals were sacrificed at either 12 days post-induction (dpi, acute stage) or 30 dpi (chronic stage) and spleen and spinal cord were collected from individual mice for further studies. Cellular infiltration and microgliosis was evaluated by IHC. Cytokine production was assessed in spinal cord and in cultured splenocytes after MOG activation ex-vivo by cytometry and real time RT-PCR. Results: Our results showed that MeCP2 deficiency increased the susceptibility to develop EAE, along with a defective induction of anti-inflammatory responses and an exacerbated MOG-specific reactivity with high IFNγ expression in peripheral immune sites. During the chronic stage, an increase in gene expression of pro-inflammatory cytokines (IFNγ, TNFα and IL-1β) and downregulation of genes relevant for immune regulation (IL-10, FoxP3 and CX3CR1) was found in MUT-EAE spinal cords. Conclusions: This is the first study performed in a MeCP2 mutant mouse model that explores the pathophysiology and neuroinflammation in the context of an autoimmune challenge. We could establish that an MeCP2 mutation act intrinsically affecting neuroimmune interactions by promoting an inflammatory environment and a deficient immune regulatory setting. These results are relevant for understanding the consequences of MeCP2 mutations on immune homeostasis in MeCP2-related disorders, as well as setting the bases for further therapeutic interventions that consider the immune status in patients.

Methyl cytosine binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Mutations in the gene encoding MeCP2 result in Rett Syndrome (RTT), a pervasive neurodevelopmental disorder. RTT is one of few autism spectrum disorders whose cause was identified as a single gene mutation. Remarkably, abnormal levels of MeCP2 have been associated to other neurodevelopmental disorders, as well as neuropsychiatric disorders. Therefore, many studies have been oriented to investigate the role of MeCP2 in the nervous system. In the present work, we explore cellular and molecular mechanisms affecting synaptic plasticity events in vivo in the hippocampus of MeCP2 mutant mice. While most studies addressed postsynaptic defects in the absence of MeCP2, we took advantage of an in vivo activity-paradigm (seizures), two models of MeCP2 deficiency, and neurobiological assays to reveal novel defects in presynaptic structural plasticity in the hippocampus in RTT rodent models. These approaches allowed us to determine that MeCP2 mutations alter presynaptic components, i.e., disrupts the plastic response of mossy fibers to synaptic activity and results in reduced axonal growth which is correlated with imbalanced trophic and guidance support, associated with aberrant expression of brain-derived neurotrophic factor and semaphorin 3F. Our results also revealed that adult-born granule cells recapitulate maturational defects that have been only shown at early postnatal ages. As these cells do not mature timely, they may not integrate properly into the adult hippocampal circuitry. Finally, we performed a hippocampal-dependent test that revealed defective spatial memory in these mice. Altogether, our studies establish a model that allows us to evaluate the effect of the manipulation of specific pathways involved in axonal guidance, synaptogenesis, or maturation in specific circuits and correlate it with changes in behavior. Understanding the mechanisms underlying the neuronal compromise caused by mutations in MeCP2 could provide information on the pathogenic mechanism of autistic spectrum disorders and improve our understanding of brain development and molecular basis of behavior.

Rett syndrome (RTT) is an autism spectrum disorder (ASD) caused by mutations in the X-linked MECP2 gene that encodes methyl-CpG binding protein 2 (MeCP2). Symptoms range in severity and include psychomotor disabilities, seizures, ataxia, and intellectual disability. Symptom onset is between 6-18 months of age, a critical period of brain development that is highly energy-dependent. Notably, patients with RTT have evidence of mitochondrial dysfunction, as well as abnormal levels of the adipokines leptin and adiponectin, suggesting overall metabolic imbalance. We hypothesized that one contributor to RTT symptoms is energy deficiency due to defective nutrient substrate utilization by the TCA cycle. This energy deficit would lead to a metabolic imbalance, but would be treatable by providing anaplerotic substrates to the TCA cycle to enhance energy production. We show that dietary therapy with triheptanoin significantly increased longevity and improved motor function and social interaction in male mice hemizygous for Mecp2 knockout. Anaplerotic therapy in Mecp2 knockout mice also improved indicators of impaired substrate utilization, decreased adiposity, increased glucose tolerance and insulin sensitivity, decreased serum leptin and insulin, and improved mitochondrial morphology in skeletal muscle. Untargeted metabolomics of liver and skeletal muscle revealed increases in levels of TCA cycle intermediates with triheptanoin diet, as well as normalizations of glucose and fatty acid biochemical pathways consistent with the improved metabolic phenotype in Mecp2 knockout mice on triheptanoin. These results suggest that an approach using dietary supplementation with anaplerotic substrate is effective in improving symptoms and metabolic health in RTT.

Experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many of the clinical and pathological features of the human disease multiple sclerosis (MS). Both are inflammatory demyelinating and neurodegenerative pathologies of the central nervous system associated with motor, sensory, and cognitive deficits. In MS, gray matter atrophy is related to the emergence of cognitive deficits and contributes to clinical progression. In particular, prefrontal cortex injury and dysfunction have been correlated to the development of fatigue, one of the most common and disabling symptoms in MS. However, the molecular bases of these changes remain unknown. Taking advantage of EAE similitude, we herein analyze functional and morphological changes in isolated cortical presynaptic terminals (synaptosomes) from an acute rat model. We found impaired glutamate release in the frontal cortex from EAE rats. This defect appeared along with the onset of the disease, reversing when clinical signs were no more evident. Biochemical analysis of EAE synaptosomes revealed alterations in the presynaptic release machinery and in the response to depolarization, which was accompanied by abnormal synapsin I phosphorylation and dispersion. These changes were associated with reduced synaptic vesicle mobility, with no alterations in synaptosomal morphology as evidenced by electron microscopy. The present are the first pieces of evidence unraveling the molecular mechanisms of frontal cortex neuronal dysfunction in EAE and, possibly, MS.

We have previously described that antibodies and T cells against myelin basic protein (MBP) rose under conditions to induce acute experimental autoimmune encephalomyelitis (EAE) bind other proteins present in the synaptosomal fraction, some of them identified as synapsin I. The aim of this study was to evaluate whether anti-MBP T-cell lines can be also activated by synapsin. The analysis of rat anti-MBP T-cell lines cultured with each antigen showed that these cells responded also to purified rat synapsin and to the amino terminal portion of this protein. This recognition originated a proliferative response with a concomitant pattern of cytokine secretion similar to that induced by MBP itself implicating that this recognition would be mediated by the T-cell receptor. On the other hand, anti-synapsin T-cell lines were not capable of responding to MBP stimulation. Therefore, the immunological cross-reactivity between both proteins occurs only in one direction and these cross-reactive cells would be elicited only in animals sensitized with MBP. A possible implication of immunological agents against MBP cross-reactive with extra-myelin proteins in the process of EAE is considered.