Introduction
The solute carrier (SLC) group of membrane transport proteins include about 400 members organized into more than 50 families (Hediger et al.
2013). SLCs mediate the selective transport of molecules such as nucleotides, amino acids, and sugars across biological membranes. However, for many of the SLC family members, it is unknown what they transport and there is a lack in our knowledge concerning the roles of different SLCs in different cellular compartments (Ziegler et al.
2021). For some SLC family members, there are some data shedding light on their possible roles.
For instance, members of the SLC25 family provide transport steps for substances across the mitochondrial inner membrane, and within that family SLC25A39 has been identified as a mitochondrial membrane carrier regulating glutathione transport into mitochondria (Wang et al.
2021). In flies, missense mutations of the SLC25A39 homolog “shawn” result in accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, synaptic defects and neurodegeneration (Slabbaert et al.
2016). Since Slc25a39 protein is expressed in the postnatal brain, malfunction of SLC25A39 might interfere with synaptic dysfunction or even neurodegeneration in mammals (von Bohlen und Halbach
2022).
Other members of the SLC superfamily seem to play a role in neurodevelopmental disorders. For example, it has recently been described that mutations in an SLC32 family member (SLC32A1) can cause developmental and epileptic encephalopathy (Platzer et al.
2022). Comparable to this, biallelic variants in SLC38A3 can cause epileptic encephalopathy (Marafi et al.
2022). SLC32A1 is also known as “vesicular γ-aminobutyric acid (GABA) transporter” (VGAT), which is involved in GABAergic neurotransmission (Platzer et al.
2022). SLC32A1 is widely expressed throughout the whole brain (see, e.g.,
http://mouse.brain-map.org/experiment/show/72081554). SLC38A3, on the other hand, encodes a glutamine transporter that is also widely expressed in the postnatal brain (
http://mouse.brain-map.org/experiment/show/72081554).
A further member of the SLC superfamily that is expressed in the human brain is SLC35F1 (Nishimura et al.
2009). SLC35F1 mRNA is expressed at high levels in the fetal and adult brain in humans (Nishimura et al.
2009); however, a detailed mapping of brain areas expressing SLC35F1 in the human brain has not been done yet. Little is known concerning the roles and functions of SLC35F1 in humans. In 2010, an association with resting heart rate at loci 6q22 near SLC35F1 has been identified (Eijgelsheim et al.
2010). In 2017, it has been described that SLC35F1 might play a role in several cardiovascular diseases based on electrocardiographic QT variations (Avery et al.
2017). Moreover, differentially methylated or expressed SLC35F1 might serve as an epigenetic biomarker for colon cancer (Wu et al.
2020). Deletions in a chromosomal region including the regulatory sequences of SLC35F1 (6q22.1q22.31) are associated with pediatric epilepsy (Szafranski et al.
2015), suggesting a neurodevelopmental role for the SLC35F1 gene. In addition, it has recently been described that a patient carrying a mutation in the SLC35F1 gene exhibited a Rett syndrome-like phenotype (RTT) (Di Fede et al.
2021): The patient, among others, experienced seizures and was unable to walk independently (Di Fede et al.
2021). Moreover, the patient displayed intellectual disability (ID; Di Fede et al.
2021).
RTT can be caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MeCP2). Mecp2-mutant mice are used in preclinical studies that target the MeCP2 gene directly, or its downstream pathways (Vashi and Justice
2019; Gonzalez-Sulser
2020). The RTT mice, which reproduce many aspects seen in the Rett syndrome, also show clear deficits in hippocampus-dependent learning and memory and hippocampal synaptic plasticity (Castro et al.
2014; Moretti et al.
2006; De Filippis et al.
2014).
For getting a better insight into the possible roles of Slc35f1 in the brain, we generated Slc35f1-deficient mice. These mice were viable and survived into adulthood. This enables us to analyze these mice on the behavioral and neuroanatomical level. We focused on the limbic system, which allows comparing the obtained data with those available from different analysis of Mecp2 mutant mice.
Discussion
Within the postnatal brain, Slc35f1 is not homogeneously distributed, but enriched in neurons located, e.g., within the amygdala, hippocampus, and cortex (Farenholtz et al.
2019). Based on the localization of Slc35f1 in the cortex and in the limbic system, Slc35f1 may be involved in neuronal plasticity or play a critical role in the maintenance of the neuronal circuitries. Slc35f1 co-localizes with Rab11 (Farenholtz et al.
2019). Rab 11 is important for dendritic spine formation, and mutations in Rab 11 have been associated with encephalopathy (Hamdan et al.
2017). To get insight into the possible roles of Slc35f1, we generated Slc35f1-deficient mice. These Slc35f1-deficient mice are fertile and show normal development. Postnatal Slc35f1-deficient mice display no seizure-like events and do not show major movement restrictions. In the OF, the mice also did not show major deficits, with the exception that 5-months old Slc35f1-deficient mice display significant reductions in velocity and traveled distance. This behavior was somewhat unexpected, since we have hypothesized that the Slc35f1-deficient mice would show severe movement restriction. This hypothesis was based on the fact that a human patient carrying a heterozygous SLC35F1 deficiency was unable to walk independently (Di Fede et al.
2021).
Moreover, since SLC35F1 deficiency can lead to a phenotype that resembles Rett syndrome (Di Fede et al.
2021), we analyzed the Slc35f1 mice in the marble-burying test. This test is commonly used to describe phenotypes in mouse models of neurodevelopmental and psychiatric disorders (Wahl et al.
2022). For instance, hemizygous MeCP2-308 mice (a mouse RTT- model) display decreased digging and buried a lower number of marbles compared their wild-type littermates (De Filippis et al.
2014). However, a comparable behavior was not seen in the Slc35f1-deficient mice, indicating that the environment-directed exploratory behavior was not altered. The MeCP2-308 mice also display altered dark–light box behavior; they spent significantly less time in the light compartment in the light/dark test compared with wild-type controls. Such a behavior was not evident in the Slc35f1-deficient mice.
Based on the fact that mutation in the SLC35F1 gene can induce severe ID in humans (Di Fede et al.
2021), we tested the Slc35f1 mice in two behavioral tests that are known to be specific for testing learning and memory. In the NOR test, learning and memory can be tested. In addition, this test has been shown to be sensitive for neuropsychological changes (Lueptow
2017). In the MWM-Test spatial learning and memory is tested (D'Hooge and De Deyn
2001). In contrast to what we expected, Slc35f1 did not show significantly altered learning and memory as compared to their age-matched controls. This may indicate (i) that in mice, deficiency for Slc35f1 might be compensated or (ii) that Slc35f1 gained importance during the mammalian brain evolution. At least in mice, deletion of Slc35f1 does not affect learning and memory, nor does it have a major impact on brain morphology. Moreover, morphological readouts of neuronal plasticity that are known to correlate with learning and memory are not altered. Adult hippocampal neurogenesis is closely linked to learning and memory that involves the hippocampus (Kempermann
2008) and adult hippocampal neurogenesis is affected in ID (Pons-Espinal et al.
2013), and in mouse models of RTT, disturbances in adult hippocampal neurogenesis have been described (Pons-Espinal et al.
2013). However, adult Slc35f1-deficient mice did not display any obvious alteration in adult hippocampal neurogenesis, neither in the capacity of generating new proliferating cells, nor in the ability to generate new neurons. However, the most obvious morphological alteration in the brain architecture in relation to ID are changes on the level of dendritic spines (von Bohlen und Halbach
2010). The analysis of dendritic spines, however, revealed only a slight reduction in the length of dendritic spines in the dentate gyrus. While missense mutation in the SLC35F1 gene in humans has disastrous effects on the brain leading to ID, deficiency of Slc35f1 has only a very mild impact on the architecture of the mouse brain and learning and memory.
Compared to the human cerebral cortex, the cortex of a mouse has more than 1000-fold smaller areas and numbers of neurons (Hodge et al.
2019). Although the basic architecture appears to be conserved, there are differences in the cellular makeup of the cortex in different mammals (Hodge et al.
2019). These differences are not only obvious on the neuronal level, but also on the level of glia cells (Yu and Zecevic
2011). Interestingly, astrocytes in the murine brain seem mainly to be negative for Slc35f1, as determined by immunohistochemistry (Farenholtz et al.
2019). In contrast, data, based on single cell RNA sequencing, hint that SLC35F1 can be detected in astrocytes (
https://www.proteinatlas.org/ENSG00000196376-SLC35F1), derived from human tissue. Recent evidences suggest that astrocytes play a role in ID (Cresto et al.
2019) and dysfunctional astrocytes may contribute to memory deficits (Fernandez-Blanco and Dierssen
2020). RTT is caused by mutations in MeCP2 (Vashi and Justice
2019), and in both mouse and human MeCP2-deficient astrocytes altered vesicular transport and microtubule dynamics have been observed (Delepine et al.
2016). Since mouse and human astrocytes seem to differ in their expression of Slc35f1, deletion of Slc35f1 has no effect on murine astrocytes, but an impact on human astrocytes, which may contribute to the phenotype of the patient carrying a mutation in the SLC35F1 gene as described by Di Fede and collegues (
2021). Thus, there are limitations in the extrapolations we can make from mouse models. At least the Slc35f1 mouse model is neither suitable to mimic the effects that result from a missense mutation of SLC35F1 in humans, nor is it a suitable animal model of RTT.
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