RBPs are integral to RNA metabolism, regulating critical functions such as splicing, transport, transcription, and translation. Dysfunctions in RBPs can lead to cellular damage, including issues such as nucleocytoplasmic mislocalization and altered protein expression. It is hypothesized that the initial event or trigger‐inducing RBP dysfunction can initiate a cascade of alterations in RNA metabolism, which can negatively impact cellular function. This highlights the importance of maintaining proper RBP function to ensure optimal cellular health. The present study demonstrates that the inhibition of hnRNP A1 splicing activity during remyelination after CUP ‐induced demyelination leads to significant alterations in the expression of essential proteins involved in myelination, such as MOG, MAG, MBP, CNP, and PLP, with a more pronounced effect observed in the PFC when compared to the CC and HPC.

CUP has been widely employed as a model for studying demyelination and remyelination in the CNS. The histopathological features associated with CUP exposure include OL death and subsequent demyelination in the CC, cortex, HPC, cerebellum, and various other regions of the mouse brain (reviewed in Vega‐Riquer et al. 2019). In the CC, 5–6 weeks after CUP exposure is withdrawn, white matter remyelination occurs; however, in cortical gray matter, remyelination does not occur as efficiently within the same timeframe (Wergeland et al. 2012). This difference in remyelination dynamics between white and gray matter was substantiated by our results. Animals exposed to CUP for 5 weeks followed by 5 weeks of remyelination displayed few commonly dysregulated proteins among brain regions. Nonetheless, some similarities were observed between the CC and PFC, such as stearoyl‐CoA desaturase 2, alpha‐crystallin B chain, and reticulon‐4. However, in contrast, a prominent downregulation of PLP persists in the PFC. The differing dynamics of remyelination between white and gray matter highlight the complexity of myelination processes in distinct brain regions and may have implications for understanding the underlying mechanisms and potential therapeutic strategies for promoting remyelination in different areas of the brain.

Consistent with the expectations of the CUP model, we observed behavioral changes, including short‐ and long‐term cognitive impairment, triggered by CUP exposure during 5 weeks (Serra‐de‐Oliveira et al. 2015; Sen et al. 2019). Although our study identified altered velocity parameters in the CUP group at 5 weeks, these animals did not exhibit changes in total distance traveled or time spent exploring objects, suggesting a specific impairment in movement coordination rather than overall locomotion.

Myelin consists mostly of lipids, which make up 70%–80% of its dry weight. The most abundant lipids in myelin sheath are cholesterol and ethanolamine plasmalogen. Proteins constitute the other approximately 20%–30% of its content (Norton and Poduslo 1973). Extracellular compaction of CNS myelin is accomplished by proteins MOG and MAG, both members of the immunoglobulin superfamily. Compact myelin predominantly comprises two significant proteins, MBP and PLP, which significantly enhance myelin membrane adhesion and ensure its stability (Lee et al. 2014; Bakhti et al. 2013). In contrast to MOG and MAG, CNP is crucial in preventing myelin compaction through cytoskeletal reorganization. The proper balance among these proteins is paramount in maintaining the appropriate level of compaction and stabilizing myelin (Domingues et al. 2018; Snaidero et al. 2017).

Several hnRNPs affect myelin mRNA post‐transcriptional processes, including the synthesis, transportation, and translation of MBP, as well as the alternative splicing of PLP (Wang et al. 2012; Ainger et al. 1997; Hoek et al. 1998; Torvund‐Jensen et al. 2014; Laursen, Chan, and Ffrench‐Constant 2011). PLP and DM20 are two protein isoforms generated through alternative splicing of the PLP gene. PLP is exclusively expressed in myelin and has distinctive signaling functions crucial for maintaining axonal integrity, primarily attributed to the inclusion of Exon 3B (Timsit et al. 1992). In the mammalian postnatal brain, the PLP protein isoform is more prevalent than DM20, and the inclusion of Exon 3B confers unique signaling functions to PLP, which play a critical role in maintaining axonal integrity through axo‐glial interactions (Hobson et al. 2006; Stecca et al. 2000). Throughout the course of OL maturation, the ratio of PLP to DM20 increases alongside a reduction in the expression of hnRNP H and F. Knockdown of hnRNP H resulted in an elevated PLP/DM20 ratio, with no effect by hnRNP F knockdown. Interestingly, the combined silencing of both hnRNP H and F exhibited a synergistic effect, leading to a more substantial increase in the PLP/DM20 ratio compared to the impact observed by hnRNP H knockdown alone (Wang, Dimova, and Cambi 2007). hnRNP A1 knockdown is also detrimental to OLs, inducing apoptosis and necroptosis (Jahanbazi Jahan‐Abad et al. 2023). In the absence of the inhibitor VPC, a downregulation of PLP is observed in the PFC. Furthermore, alterations in PLP expression continue to occur even in the presence of the inhibitor, and additional key myelin proteins are also affected not only in the PFC but also in the CC and HPC. These findings underscore the substantial role of hnRNP A1 in regulating the expression of myelin proteins and may explain the differences in the remyelination process among brain regions.

Further supporting this idea, expression levels of proteins involved in the sphingolipid signaling pathway were altered, including acid ceramidase (ACDase), tyrosine‐protein kinase Fyn (FYN), cathepsin D (CTSD), and MAPK family proteins. ACDase breaks down sphingolipids in lysosomes by converting ceramide into sphingosine and a free fatty acid. A deficiency in ACDase results in a wide array of alterations in the CNS (Sikora et al. 2017). FYN, a crucial protein for CNS myelination, activates signaling pathways necessary for both the initial stages of brain development and myelin regeneration in chronic demyelinating diseases (Baer et al. 2009; Sperber et al. 2001). Mutations in the Ctsd gene can cause severe neurological diseases and lead to a loss of neurons, myelin, and gliosis. Ctsd‐knockout mice also exhibit significant myelin changes in the brain, suggesting that CTSD plays an essential role in CNS myelination by influencing the trafficking of PLP (Fritchie et al. 2009; Trajkovic et al. 2006; Mutka et al. 2010; Guo et al. 2018).

The process of myelin formation in the CNS is a complex one, involving several intricate steps. First, OL precursor cells that have proliferated must migrate to their final location within the brain. Upon reaching this stage, they differentiate into postmitotic OLs that will myelinate multiple axons. Any anomaly in these processes can severely compromise the myelination process (reviewed by Gouvêa‐Junqueira et al. 2020). Our findings highlighted the crucial role that hnRNP A1 splicing activity plays in regulating these steps, thereby underscoring the need for further, in‐depth investigations into the potential of modulators of hnRNP A1 splicing activity as a therapeutic target for demyelinating disorders.

Endocannabinoids function as retrograde messengers within the CNS, regulating synaptic function and plasticity (Alger 2012). They are involved in various cognitive and physiological processes, such as learning, memory, feeding behavior, stress response, and reward (Kruk‐Slomka et al. 2017; Morena et al. 2016; Sagheddu et al. 2015; Bermudez‐Silva et al. 2010). These messengers also play a role in synaptic maturation and neurogenesis during development (Harkany et al. 2007). Dysregulation of endocannabinoid signaling is linked to psychiatric, neurological, and neurodevelopmental disorders such as anxiety, depression, epilepsy, Alzheimer's disease, Huntington's disease, and autism (Cristino, Bisogno, and Di Marzo 2020). There are variations in proteins that play a significant role in retrograde endocannabinoid signaling in all three brain regions that were studied; however, there is limited similarity in the individual proteins that are shared among regions. The dysregulation of the endocannabinoid system in the PFC impacts various cognitive abilities, including decision‐making, attention, motivation, and controlling emotions. In the HPC, dysfunctions can lead to difficulties in learning and memory, as well as affect emotional balance and stress responses (Liu et al. 2017).

Fatty acid amide hydrolase (FAAH), an enzyme responsible for metabolizing the endocannabinoid anandamide in postsynaptic neurons, was found to be dysregulated in the hippocampal proteome. FAAH overexpression at the postsynaptic site of CA1‐CA3 neurons has been associated with an increase in anxiety‐like behavior, and deficits in both object recognition memory and the extinction of aversive memory (Zimmermann et al. 2019). Although the use of the inhibitor VPC during the remyelination period did not impact the evaluated behaviors, the observed alteration in FAAH expression suggests a potential role for hnRNP A1 splicing in regulating FAAH expression, particularly in the context of myelination processes. Notably, high FAAH levels, which increase with OLs maturation, were found in OLs of the cortex and spinal cord (Moreno‐Luna et al. 2020). This modulation of FAAH expression, triggered by the inhibition of hnRNP A1 activity, likely reflects an adaptive response to the remyelination process. Many neuronal processes, such as axogenesis, synaptic maturation, and long‐term potentiation, are regulated by hnRNPs (Folci et al. 2014; Kim et al. 2013; Proepper et al. 2011). Indeed, exposure to an inhibitor of hnRNP A1 altered the expression of proteins involved in the synaptic vesicle cycle in the CC and PFC. More specifically, neural‐specific syntaxin‐binding protein 1, a regulator of synaptic vesicle docking and fusion (Pevsner, Hsu, and Scheller 1994), was dysregulated in both regions. The PFC alone exhibited additional changes, such as in adaptor protein complex 2, associated with clathrin‐mediated endocytosis. Moreover, modifications were noted in calmodulin components, which were identified in preparations of presynaptic cytoplasm and highly enriched synaptic vesicle fractions. Calmodulin also regulates the phosphorylation of several proteins in response to Ca²⁺ in fractions, such as synaptic vesicles, synaptic membranes, synaptic junctions, and postsynaptic densities (Collins et al. 2002; Xue et al. 2021). As such, the inhibition of hnRNP A1 splicing activity could potentially impact the function of synaptic vesicles in a remyelination context.

Although we did not find any evidence of impaired long‐ or short‐term memory in the mice exposed to the VPC inhibitor during the remyelination period, we observed alterations in the expression levels of proteins related to GABAergic synapses in the HPC and CC, as well as dopaminergic synapses and long‐term potentiation in the PFC. These alterations could potentially be linked to the modifications in endocannabinoid signaling that were observed within these same regions.

Information storage in long‐term memory is believed to involve changes to the relevant synapses. In mammals, the major experimental model for memory formation at the synaptic level is long‐term potentiation (Bliss and Collingridge 1993). The model's validity relies on similarities between cellular mechanisms in HPC ‐dependent learning and hippocampal long‐term potentiation, particularly their reliance on NMDA receptors (Tsien, Huerta, and Tonegawa 1996). There is increasing evidence that GABAergic interneurons have a significant impact on the ability of hippocampal principal neurons to undergo synaptic changes during learning (Paulsen and Moser 1998). Our analysis revealed alterations in the expression levels of key proteins for GABAergic synapses in the HPC, such as protein kinase C gamma type (PKC), Na(+)/Cl(−) betaine/GABA transporter (BGT‐1), and glutamate decarboxylase 2 (GAD65). PKC is a kinase with broad activity that participates in multiple signaling pathways in the brain. By phosphorylating postsynaptic transporters such as NMDA‐type glutamate receptors and GABA receptors, PKC plays a vital role in regulating synaptic plasticity (Bright and Smart 2013; Foster and Vaughan 2017). Moreover, PKC–mediated phosphorylation of GluA2 subunits of AMPARs is necessary for the internalization of AMPARs from the postsynaptic membrane, which promotes long‐term depression (Boehm et al. 2006). The enzyme responsible for GABA synthesis and regulation of vesicular release in GABAergic neurons, GAD65, was found to be upregulated in the HPC. Other GABAergic synapse proteins were also dysregulated in the CC, specifically glutamate decarboxylase 1 (GAD67) and glutamine synthetase. GAD67, a component of the GABA‐A receptor, plays a significant role in neurotransmission by synthesizing cytoplasmic GABA, whereas glutamine synthetase converts glutamate to glutamine. Glutamine synthetase is exclusively expressed in glial cells, but its product can be transferred to neurons, where it can be reconverted to glutamate, serving as a precursor of GABA. This process significantly contributes to regulating the glutamine–glutamate–GABA cycle (Grone and Maruska 2016; Walls et al. 2015). These findings provide additional evidence of multiple alterations in the GABAergic system following the inhibition of hnRNP A1 splicing activity. Although no behavioral impairments were observed in the animal model, molecular changes seemed to underlie alterations at the cellular and synaptic levels. Specifically, it appears that hnRNP A1 splicing can modulate the GABAergic system, but other molecular changes may also be required before a phenotypic alteration can be observed.

Potential limitations of the present study are: (1) we exclusively utilized male subjects, and therefore, it would be useful to expand the investigation to include female subjects to determine any potential sex‐specific differences; and (2) since hnRNP A1 is present in various cell types, the observed alterations may not be exclusively attributed to impaired OLs, but could involve other neuronal cell types such as neurons, astrocytes, and microglia, thereby warranting further exploration. Addressing these limitations would contribute to a more comprehensive understanding of the role of hnRNP A1 in neurobiology and pathology.