Characterization of lncRNA Profiles of Plasma-Derived Exosomes From Type 1 Diabetes Mellitus

Backgrounds Exosomes contain several types of transcripts, including long non-coding RNAs (lncRNAs), and have been shown to exert important effects in human diseases. However, the roles of exosomal lncRNAs in type 1 diabetes mellitus (T1DM) have not been well investigated. In the present study, we characterized the plasma-derived exosomal lncRNAs expression profiles of T1DM and predict their potential function in the pathogenesis of T1DM. Material and Methods Exosomal lncRNA expression profiles were detected by Illumina Hiseq platform (T1DM subjects N=10; age-, sex- matched Control subjects N=10). Six exosomal lncRNAs were selected to validate their expression level by using quantitative real-time PCR (qRT-PCR) (T1DM subjects N=30; age-, sex- matched Control subjects N=30). Bioinformatics analysis approaches were carried out to explore the potential biological function of differentially expressed lncRNAs. Results A total of 162 differentially expressed exosomal lncRNAs were identified in T1DM patients compared with control subjects, among which 77 up-regulated and 85 down-regulated. The expression level of the selected six lncRNAs didn’t show significant difference in the following qRT-PCR analysis. Gene Ontology analysis enriched terms such as activation of phospholipase D activity, neuronal cell body membrane, and calcium sensitive guanylate cyclase activator activity for cis-acting genes of lncRNAs, and metal ion binding for trans-acting genes. The most enriched Kyoto Encyclopedia of Genes and Genomes pathways for the lncRNAs were associated with oxidative phosphorylation and Parkinson’s disease for cis-acting genes, and pathways in cancer as well as focal adhesion for trans-acting genes. Conclusions This study characterized the lncRNA profiles of plasma-derived exosomes from T1DM for the first time and these results highlighted the potential role of exosomal lncRNAs in T1DM pathogenesis. A better understanding of exosomal lncRNA profiling will provide novel insights into its molecular mechanisms.

INTRODUCTION
Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by absolute insulin deficiency and resultant hyperglycemia (1,2). The incidence and prevalence of T1DM are increasing worldwide and more than 463 million people are affected (3,4). This disease is incurable at present and patients with T1DM have to rely on lifelong insulin administration. Besides, the high prevalence of diabetic complications, such as retinopathy, nephropathy, as well as neuropathy, severely influence the life quality of T1DM patients and impose a considerable financial burden. The pathogenesis of T1DM is extremely complex and multiple factors, including genetic backgrounds, environmental triggers, and behavioral changes, may contribute to the onset and development of T1DM (5)(6)(7). An improved understanding of T1DM pathophysiology will render early identification and intervene of T1DM.
Growing evidence has suggested that a small extracellular vesicle (EV), namely exosome, plays an important role in multiple pathogenic processes, including T1DM (8). Exosomes, which can be released by virtually all cell types, are significant mediators of intercellular communication and interorgan crosstalk through transferring bioactive molecules, including proteins, lipids, and RNAs between cells (9,10). Moreover, the content of exosomes is strictly regulated in response to all kinds of endogenous or exogenous stimulations, thus reflecting the biological events or disease conditions (11). Therefore, a better understanding of exosomes may provide a valuable target for disease diagnosis and treatment.
Long non-coding RNAs (lncRNAs), a sub-class of noncoding RNA family, have emerged as crucial regulators in multiple pathophysiological conditions (12). LncRNAs are more than 200 nucleotides in length and take part in the regulation of various cellular and biological processes, such as gene silencing, histone modifications and DNA methylation (13,14). Accumulating evidence has highlighted the role of lncRNAs in pancreatic islets and development of T1DM (15)(16)(17)(18). However, the research about the exosomal lncRNA is relatively lacking, especially in T1DM. Here, we investigated the plasmaderived exosomal lncRNA expression profiles in patients with T1DM and explored their biological function. This present work provided a novel insight into the pathogenesis of T1DM and laid a foundation for using exosomal lncRNAs as biomarkers and therapeutic targets for T1DM.

Study Subjects
Patients with T1DM attending the Diabetes Clinic at the Second Xiangya Hospital were recruited. The inclusion criteria of case group were as follows: (1) fulfilling the WHO diagnostic criteria for diabetes (1999); (2) acute onset and insulin dependency within 6 months after diagnosis; (3) positive for at least one following islet autoantibodies: GADA (glutamic acid decarboxylase antibody), IA-2A (protein tyrosine phosphatase antibody), or ZnTA8 (zinc transporter 8 antibody); (4) diabetes duration less than 5 years. Exclusion criteria included pregnancy, combined with other autoimmune diseases, malignant tumors, or a recent cardiovascular event. Non-diabetic subjects without autoimmune diseases, cancers, or family history of diabetes were recruited as health control. This case-control study was approved by the Ethics Committee of the Second Xiangya Hospital and all the research methods were conducted in accordance with the ethical guidelines of the Declaration of Helsinki. All the participants or their guardians indicated that they fully understand the research goals and procedures, and written informed consent was obtained. A total of 10 cases and 10 controls were included in the discovery phase. There was no significant difference in age (P=0.97) and sex (P=0.36) between two groups (Table 1). Besides, we used a new cohort (T1DM subjects N=30; age-, sex-matched Control subjects N=30) for the following qRT-PCR analysis.

Isolation of Plasma
Peripheral blood was collected in the EDTA blood tubes from each participant and plasma was separated by centrifugation at 3000 g for 15 min at 4°C. The plasm samples were stored at -80°C before use.

Isolation of Exosome
Size exclusion chromatography methods were adopted to isolate exosomes. In brief, 1 mL of 0.8 mm filtered plasma was 1.5-fold diluted with phosphate-buffered saline (PBS) and the further purification was made by using Exosupur ® columns (Echobiotech, China). Next, we eluted the samples with 0.1M PBS and collected about 2 mL elute fractions. The collected fractions were then concentrated to 200 mL by 100 kDa molecular weight cut-off Amicon ® Ultra spin filters (Merck, Germany). All procedures were operated according to the manufacture's institutions.

Transmission Electron Microscopy (TEM)
We put 10 µL purified exosome on a copper mesh and the sample was incubated at room temperature for 1 min . The exosome was  negatively stained with uranyl acetate solution for 1 min after  washing with sterile distilled water. After dried for 2 min under  incandescent light, the exosome was examined under a TEM (H-7650, Hitachi Ltd., Tokyo, Japan).

Nanoparticle Tracking Analysis (NTA)
The size distribution and quality of isolated particle (concentrations between 1x10 7 /mL and 1x10 9 /mL) were determined by the ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany), which equipped with 405 nm laser. A 60 second video was taken with a frame rate of 30 frames/second. The, the particle movement was analyzed by NTA software (ZetaView 8.02.28).

Western Blot Analysis (WB)
The exosome supernatant was denatured in 5 × sodium dodecyl sulfonate (SDS) buffer and subjected to WB analysis (10% SDSpolyacrylamide gel electrophoresis; 50 µg protein/lane) using rabbit polyclonal antibody CD63, TSG101, Alix and calnexin. The proteins were visualized on the Tanon4600 Automatic chemiluminescence image analysis system (Tanon, Shanghai, China). The identification of exosome, including TEM, NTA, and WB analysis, was entrusted the company (Echo Biotech Co., Ltd, Beijing, P. R. China).

Total RNA Isolation
The miRNeasy Serum/Plasma Advanced Kit (Qiagen, cat. No. 217204) was used to extract and purify the exosomal RNA in compliance with the kit instruction. The concentration and purity of RNA were evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA).

Library Preparation and Sequencing
The extracted RNA was used as input material for sequencing libraries using the SMARTer Stranded Total RNA-Seq Kit (Takara Bio USA, Inc.) according to manufacturer's recommendations. Index codes were added to attribute sequences to each sample. At last, library quality was evaluated on the Agilent Bioanalyzer 2100 and qPCR. The clustering of the index-coded samples was performed on acBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS (Illumina, San Diego, CA, USA). After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.

LncRNA Analysis
The transcriptome was assembled using the Stringtie and Scripture based on the reads mapped to the reference genome. The assembled transcripts were annotated using the Cuffcompare program from the Cufflinks package. The unknown transcripts were used to screen for putative lncRNAs. Four computational approaches including CPC, CNCI, Pfam, and CPAT were combined to elect ncRNA candidates from presumed protein-coding RNAs in the unknown transcripts. Presumed proteincoding RNAs were filtered out using exon number threshold and a minimum length. Transcripts having more than two exons and with lengths more than 200 nt were selected as lncRNA candidates and further screened using CPC, CNCI, Pfam, and CPAT that have the power to distinguish the proteincoding genes from the non-coding genes. As well as the different types of lncRNAs include long intergenic lncRNA (lincRNA), intronic lncRNA, anti-sense lncRNA were selected using cuff compare. Stringtie was used to calculate FPKMs of lncRNAs in each sample. Gene FPKMs (fragments per kilo-base of exon per million fragments) were computed by summing the FPKMs of transcripts in each gene group. The lncRNA sequencing and lncRNA analysis were entrusted the company (Echo Biotech Co., Ltd, Beijing, P. R. China).

GO and KEGG Pathway Enrichment Analysis
Gene Ontology (GO) enrichment analysis of the differentially expressed lncRNAs was implemented by the topGO R packages. We used KOBAS (19) software to test the statistical enrichment of differential expression genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (http://www.genome.jp/kegg/).

Quantitative Real-Time PCR (qRT-PCR) Assay
The total RNA from exosomes was extracted using miRNeasy Serum/Plasma Advanced Kit (Qiagen, cat. No. 217204) according to the manufacturer's protocol. The total RNA was then reverse transcribed to synthesize cDNA using PrimeScript ™ RT reagent Kit (Perfect Real Time) (TAKARA, RR037A). The abundance of target gene expression was detected by TaqMan ® probe using qRT-PCR. 2 µL of cDNA was used as the template for each PCR reaction. The GADPH was selected as the internal reference gene. The sequence of primers and probes were shown as Table 2.

Statistical Analysis
We used SPSS software version 20.0 to perform the statistical analysis. The data were presented as mean ± SD (standard deviation). Differential expression analysis of lncRNAs between two groups was performed using the Mann Whitney U test with cutoff FPKM > 5, P-value < 0.05 and Fold change > 1.5. The comparisons of the relative expression level of candidate lncRNAs in qRT-PCR were analyzed by the unpaired t-test. A P-value less than 0.05 was considered as statistically significant.

Characterization of Exosome
We used TEM and NTA to evaluate the morphology and sizes distribution of isolated exosome. As shown by Figure 1, the isolated particles were oval and cup-shaped with diameter range between 30 nm to 200 nm (median, 91.7 nm). WB analysis indicated that the presence of exosomal positive protein markers Alix, Tsg101, and CD63, and the absence of Calnexin, the negative protein marker for exosome. All these results suggested that the isolated exosomes were well-prepared and with good purity.

An Overview of the RNA Sequencing Results
The procedures of lncRNA sequencing and bioinformatics analysis were summarized in Figure 2. A total of 20 samples, including 10 patients and 10 healthy controls were constructed and analyzed. The percentage of Q30 base was at least 91.94%. A total of 40065 lncRNAs, including 33453 known lncRNAs and 6612 novel lncRNAs were generated. Among these identified lncRNAs, lincRNAs accounted for the largest proportion (69.1%), followed by intronic lncRNAs (15.5%), sense lncRNAs (8.6%), and antisense lncRNAs (6.8%). Then, we predicted the target genes of lncRNA. On the one hand, it is predicted that the adjacent genes within 100 kb were its cis-acting gene because lncRNA could regulate the expression of neighboring genes. On the other hand, the trans-acting genes of the lncRNA were predicted by analyzing the expression correlation between lncRNA and corresponding mRNA. The genes with absolute Pearson correlation coefficient greater than 0.9 and P-value less than 0.01 were taken as the trans-acting genes of the lncRNA.

Differentially Expressed Exosomal lncRNAs
Based on the plasma-derived exosomal lncRNA expression profiles, differentially expressed lncRNAs were distinguished between T1DM patients and control subjects. Hierarchical clustering analysis was performed to cluster lncRNAs according to their expression level ( Figure 3A). As shown by volcano diagram ( Figure 3B) and MA plot ( Figure 3C), total of 162 differentially expressed lncRNAs were identified, in which 77 up-regulated and 85 down-regulated ( Table 3). These results indicated that plasma-derived exosomal lncRNA expression profiles were distinguishable between T1DM patients and control subjects.

Functional Analysis of Exosomal lncRNAs
To explore the potential biological function of 162 differentially expressed lncRNAs, GO enrichment analysis and the KEGG pathway analysis were employed. The lncRNAs exert biological effects by acting as cis-or trans-regulators (20,21). We summarized the significantly enriched GO terms of cis-acting genes of differentially expressed lncRNAs regarding to the biological process (BP), cellular component (CC), and molecular function (MF), respectively ( Figure 4A). For BP, the term with the most genes and with the highest enrichment score was the activation of phospholipase D activity (GO:0031584) ( Figure 4B). For CC, the term with the most genes was the extracellular exosome (GO:0070062) and with the highest score was neuronal cell body membrane (GO:0032809) ( Figure 4C). In addition, for MF the terms DNA binding transcription factor activity (GO:0003700) and protein binding (GO:0005515) ware associated with most genes, and the most enriched term was calcium sensitive guanylate cyclase activator activity (GO:0008048) ( Figure 4D). We also summarized the GO terms of trans-acting genes ( Figure 5A). For BP, the term with the most genes was the positive regulation of transcription from RNA polymerase II promoter (GO:0045944) ( Figure 5B). For CC, the term cytosol (GO:0005829) was correlated to most genes ( Figure 5C). For MF, the term with protein binding (GO:0005515) was most genes, and metal ion binding (GO:0046872) was most significantly enriched term ( Figure 5D). Besides, the enriched pathways of target genes of lncRNA were analyzed by KEGG. For cis-acting genes, the most significantly enriched terms were involved in oxidative phosphorylation (ko00190) and Parkinson's disease (ko05012) ( Figure 6A). For trans-acting genes, the terms pathways in cancer (ko05200) and focal adhesion (ko04510) were related to most genes ( Figure 6B). We also performed the disease annotation of identified lncRNAs through sequence alignment with LncRNADisease database. The results showed some lncRNAs were associated with autoimmune diseases, including dermatomyositis and polymyositis. LncRNA can be precursor molecule of miRNA. We also detected the lncRNAs which serve as precursor of miRNA through comparing and predicting the miRNA sequence in miRbase database.

Validation of Exosomal lncRNA Expression by qRT-PCR
To further verify the sequencing data, six differentially expressed exosomal lncRNAs, including one up-regulated (ENST00000525207) and five down-regulated (ENST00000495420, ENST00000488260, ENST00000505090, ENST00000472614, and ENST00000533796), were randomly selected to perform the qRT-PCR analysis (T1DM subjects N=30; age-, sex-matched Control subjects N=30). However, the results indicated that the expression levels of selected lncRNAs were similar between T1DM patients and control individuals (Figure 7).

DISCUSSION
Nowadays, it has been reached a consensus that T1DM is a multifactorial disease, and the precise pathogenic mechanisms are still obscure. Mounting evidence has suggested exosomes play an important role in the onset and development of T1DM (8,22). However, research about the expression profiles of exosomal lncRNA in T1DM has not yet been reported. Here, we performed the transcriptome-wide expression patterns of plasma-derived exosomal lncRNAs in the T1DM using Illumina Hiseq platform, and further explore their function by bioinformatics analysis.
In the present study, 162 aberrantly expressed exosomal lncRNAs, including 77 up-regulated and 85 down-regulated, were identified. The lincRNA accounted for the most proportion of differentially expressed lncRNAs, which was in accordance with previous study (23). To further validate the expression level of identified exosomal lncRNA, six of them, one up-regulated and five down-regulated, were randomly selected to perform qRT-PCR analysis. However, no significant difference was detected between T1DM patients and control subjects.  The relatively limited sample size may contribute to the negative results more or less. Nevertheless, there were still 156 differentially expressed lncRNAs to be further test in future studies. We believe that some of these may have the potential to become novel biomarkers for T1DM given the fact that both exosome (22) and lncRNA (15) have been reported to be significantly associated with T1DM. It has been indicated that islet-derived exosomes can activate immune response and lead to autoimmune attack (24). Also, T lymphocyte-derived exosomes are associated with beta-cell dysfunction and death (25). Some studies also evaluated the diagnostic potential of exosomes in the context of T1DM. It has been demonstrated that human isletderived exosomal RNAs were differentially expressed when exposed to proinflammatory cytokines (26). Another study investigated the plasma-derived exosomes and reported a distinct exosomes miRNAs signature in patients with longduration T1DM (27). Some studies have also investigated the potential role of lncRNA in the T1DM pathogenesis. LncRNAs usually expressed in a cell-specific manner. Previous transcriptome profiling studies have identified more than 1000 islet-specific lncRNAs (28,29). The dysfunction of lncRNAs may lead to autoimmune response and alter the progression of T1DM (30,31). A recent whole genome RNA sequencing study in circulating leukocytes identified 393 differentially expressed lncRNAs, potentially providing novel targets for diagnosis and treatment of T1DM (32). Another study conducted a comparative analysis of the expression profiles of lncRNA by analyzing published microarray data set and proposed a specific 26-lncRNA signature, which could be used to effectively identify T1DM susceptible individuals (33). In addition, in vitro  experiment using MIN6 cells found that the expression of lncRNA was m od ifi ed by cytokine treatment a nd overexpression of these lncRNA favored beta-cell apoptosis, indicating dysregulation of lncRNA contributes to the beta-cell failure during the development of T1DM (18). Furthermore, some specific lncRNAs have been reported to be implicated in beta-cell function and T1DM. It has been indicated that lncRNA MALAT1 could induce beta-cell dysfunction through inhibiting the expression of PDX-1 by reducing its H3 histone acetylation (34). The lncRNA Lnc13 could regulate the inflammation of pancreatic beta-cell by allele-specific stabilization of STAT1 mRNA (35). All the aforementioned studies suggested that bo t h exo so me a nd lnc RN A w er e i nv ol ved i n t he T1DM pathogenesis. Next, to predict the potential biological functions of identified differentially expressed lncRNAs, we performed GO enrichment analysis and the KEGG pathway analysis. The terms such as activation of phospholipase D activity, neuronal cell body membrane, calcium sensitive guanylate cyclase activator activity, and metal ion binding were significantly enriched in the identified differentially exosomal lncRNAs. These terms were associated with diabetes or its complication to some extent. For instance, it has been shown that glycosylphosphatidylinositolspecific phospholipase D level were correlated to the insulin resistance of adipose tissue in obese subjects (36). Also, the expression of glycoprotein phospholipase D (GPLD1) was found to be increased in mouse models of T1DM (37,38), and GPLD1 levels in autoimmune diabetes were higher than that in T2DM or healthy controls (39).
KEGG pathway analysis indicated that the differentially expressed lncRNAs were involved in oxidative phosphorylation, Parkinson's disease (PD) and pathways in cancer. PD is also a multifactorial disease and both genetic and environmental factors contribute to the pathogenesis of this progressive neurodegenerative disease (40). The primary pathogenic mechanisms of PD remain unclear. However, some pathophysiological processes, such as mitochondrial dysfunction, oxidative stress, and chronic inflammation, are identified to be associated with PD (41). These processes are also involved in the onset and development of T1DM (42,43), which suggested that PD and T1DM might share some common pathological features. Interestingly, another progressive neurodegeneration disease, Alzheimer's disease (AD), has been proposed as "type 3 diabetes mellitus" because of the shared molecular and cellular characteristics with T1DM and T2DM (44).
LncRNAs were frequently precursor RNAs of miRNAs. We identified the precursor lncRNAs of miRNA through comparing miRNA sequence in miRbase database. Some identified miRNAs such as miR-21 (45) and miR-424 (46) has been implicated in the progression of T1DM. Therefore, the lncRNA combined miRNA might contribute to the T1DM collectively.
Both exosome and lncRNA have been implicated in the progression of T1DM. However, the exact role of exosomal lncRNA in T1DM lacks full investigation and research. The present study identified the expression profiles of exosomal lncRNA and laid foundation for further study in this field. There are some limitations of our study. First, the sample size was relatively small. Future study should include more samples to decrease the random errors. Second, we only selected six lncRNAs to validate their expression level by qRT-PCR and the sample size was relatively small. Further study should focus on more lncRNAs based on the sequencing results and assess the biomarker potential of exosomal lncRNAs in a larger sample set. Third, we didn't investigate the function of exosomal lncRNAs and some in vitro or in vivo study are required in future study.
In conclusion, this study identified the characteristics of the plasma-derived exosomal lncRNA transcriptome of T1DM for the first time and threw insights into the biomarker potential and pathogenic factor of exosomal lncRNA in T1DM.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. This data can be found here: [https://db.cngb.org/cnsa/ accession number CNP0002574].

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Xiangya Hospital. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
ZZ, ZX, and HP conceived and designed the experiments. HP, WF, JLi, YW, SL, JLin, GH, and XL collected samples. HP, XS, and WF performed the experiments and analyzed the data. HP wrote the manuscript. All authors contributed to the article and approved the submitted version.


s2fieldsofstudy: Biology Medicine

text:
Adverse Cardiac Effects of CAR T-Cell Therapy: Characteristics, Surveillance, Management, and Future Research Directions

This review summarizes the current literature on the adverse cardiac effects of CAR T-cell therapy. Case reports and series suggest that major adverse cardiovascular events are not uncommon after CAR T-cell therapy; however, limited data exist regarding incidence, pathophysiology, and prevention strategies related to CAR T-associated cardiovascular events. As cellular therapy advances and the indications for its use continue to expand, it is essential to better understand its associated cardiovascular toxicities. Biomarkers, cardiac imaging, longitudinal data from larger populations, and translational research are all essential areas for further research. Interestingly, CAR T-cell therapy can also be used to reverse cardiac fibrosis in murine models. Altogether this underscores the need to broadly understand how T-cells, endogenous and engineered, may impact cardiovascular diseases.

Introduction
Immunotherapy is now a well-established pillar of cancer therapy in addition to standard chemotherapy, surgery, and radiation. The discovery of tumor-mediated immunosuppression and its relationship with malignancy progression paved the way for T cell-activating strategies. The success of checkpoint inhibitors led to a paradigm shift in oncology and direct therapeutic use of T-cells for therapy is now quickly evolving.
Chimeric antigen receptor T-cells (CAR T-cells) are T lymphocytes that have been genetically engineered to produce a specific T-cell receptor. Chimeric antigen receptors (CARs) have undergone many iterations of stepwise advancement and were first approved by the Food and Drug Administration (FDA) in 2017 for relapsed/refractory B-cell Acute Lymphoblastic Leukemia (B-ALL) in children and young adults, and diffuse large B-cell lymphoma (DLBCL) in adults. 1 In particular the unique advantage of this system is that unlike innate T cells, CAR T-cells can recognize antigens without a presentation from the major histocompatibility complex (MHC). Furthermore, the chimeric receptor combines both antigen-binding and T-cell activating functions to allow for immune activation, tumor cell recognition, and destruction. Essentially T cells are isolated from either a patient's own blood (autologous) or from a healthy donor (allogeneic), genetically engineered to express a specific CAR, and then infused into the patient. 2 Prior to infusion patients often receive a lymphodepleting chemotherapy regimen most commonly including fludarabine and cyclophosphamide. 3 This promotes selective expansion of the adoptively transferred T-cells over endogenous lymphocytes. The target antigen is relatively specific to cancer cells to avoid the destruction of healthy cells and as the CAR T-cells detect their target antigen they become further activated and proliferate in vivo in order to treat malignancy ( Figure 1).
Other types of T-cell therapies are also currently being developed, including bispecific T-cell engager (BiTE) antibodies. BiTE therapies are synthetic antibodies that bind two different antigens: one is a tumor-specific antigen and the other is an antigen that allows binding to endogenous immune cells. This simultaneous binding of T-cells and tumor cells allows for more efficient tumor cell lysis. Compared to CAR T-cell therapy which requires successful leukapheresis, T-cell isolation, viral transduction, and patient conditioning, BiTE therapy can be available off the shelf and does not require lymphodepleting chemotherapy (Table 1). 3,4 Blinatumomab is the first BiTE antibody to be FDA approved. It redirects CD3 + T-cells to CD19 + leukemic blasts and is approved for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and BCP-ALL with minimal residual disease. 3 A single-arm trial blinatumomab showed a complete remission rate of 32% and the median duration of response was 6.7 months. 5 CAR T-cells are given typically as a single infusion and the T-cells can persist and expand in patients. However, blinatumomab has an in vivo half-life of 2-4 hours and requires continuous intravenous infusion over 28 days per cycle. 4 The benefit of this approach is however that the infusion can be stopped at any time to reverse immune activation and immune-related adverse events. On the other hand, CAR T-cells have the potential to engraft long-term and create a constant pool of T cells that can quickly respond to disease recurrence before it becomes clinically evident.  As CAR T-cells become activated, proliferate, and carry out their cytotoxic functions there can often be a significant release of inflammatory cytokines that cause known side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Severe CRS has been associated with a higher disease burden and is typically treated with supportive care, corticosteroids, and tocilizumab (anti-IL6 monoclonal antibody). 6 In this review, we aim to summarize existing evidence on the monitoring, detection, and management of cardiac injury among patients receiving cellular therapies and provide areas of need for future research.
Axicabtagene ciloleucel (axi-cel) is approved for relapsed/ refractory DLBCL and follicular lymphoma in adults. 13 In the initial phase 2, multicenter study axi-cel showed an overall response rate of 82%, complete response rate of 54% at 6 months, and an overall survival rate of 52% at 18 months. 14 Brexucabtagene autoleucel is approved for relapsed/refractory mantle cell lymphoma (MCL) and B-ALL. Its phase 2 study showed an overall response rate of 93%, a complete response rate of 67% at 6 months, and an overall survival rate of 83% at 12 months. 15 Tisagenlecleucel is approved for relapsed/ refractory B-cell ALL in children and young adults and also for relapsed/refractory DLBCL in adults. In its phase 2 study for relapsed/refractory B-cell ALL there was an overall survival rate of 76% at 12 months. 16 In another phase 2 study for relapsed/refractory DLBCL there was an overall response rate of 52%, complete response rate of 40%, and a relapse-free survival rate of 65% at 12 months. 17 Lisocabtagene maraleucel is approved for relapsed/refractory large B-cell lymphoma. Its phase 1 study showed an overall response rate of 73%, a complete response rate of 53%, and an overall survival rate of 58% at 12 months. 18 Idecabtagene vicleucel is approved for relapsed/ refractory multiple myeloma. 19,20 Its phase 2 study showed an overall response rate of 73%, a complete response rate of 33%, and an overall survival rate of 78% at 12 months. 20 Ciltacabtagene autoleucel is approved for relapsed/refractory multiple myeloma and its phase 1b/2 study showed an overall response rate of 97%, a complete response rate of 67%, and an overall survival rate of 80% at 12 months. 21 Newer research is constantly building on the CAR T-cell concept to target additional malignancies including solid tumors and even expanding the concept into non-malignant diseases such as autoimmune diseases. 22,23 As indications and applications of CAR T-cell therapy continue to expand, it is crucial to better understand treatment-associated toxicities and how to prognosticate and manage them effectively. Conversely, given recent experimental findings suggesting a potential role of certain CAR T-cell approaches in reversing cardiac fibrosis, it is likewise essential to learn from current CAR T-cell approaches to inform the potential development of cardiovascular immunotherapeutics. 24

Adverse Cardiac Effects
Cardiovascular toxicities are not uncommon among patients undergoing CAR T-cell therapy. 25 A recent pharmacovigilance study of the FDA adverse event reporting system reported 2.8% tachyarrhythmias, 2.6% cardiomyopathies, 1.8% cardiogenic  27 Age greater than 60 years, earlier CRS start, CRS grade greater than or equal to 3, longer duration of CRS, and need for tocilizumab were all significantly associated with increased thirty-day risk of MACE. 27 Alvi et al also reported that of 137 patients studied, 55 patients experienced CRS grade 2 or higher and within that cohort 24 patients had a positive troponin after treatment. Among patients with a positive troponin after treatment, eight patients had newly reduced ejection fraction. In this study, five patients also experienced new arrhythmias and six patients experienced cardiovascular death. 28 Lefebvre et al showed that approximately 21% of patients experienced a MACE. With regards to arrhythmias, tachyarrhythmias are more common than bradyarrhythmias, and Goldman et al reported atrial fibrillation to be more common than ventricular arrhythmias. 26 Not many studies have examined the incidence of electrocardiogram (ECG) changes; however, a study of cardiac dysfunction among pediatric CAR T-cell recipients showed that 18% of patients with hypotension requiring inotropic support had new ST segment changes. 29 Other studies have also reported asymptomatic prolongation of the QTc interval 30 and new-onset supraventricular tachycardia, and atrial fibrillation or flutter. 28,31 Furthermore, chest pain has not been consistently studied but the FDA package insert for ciltacabtagene autoleucel does report chest pain as an adverse event occurring in approximately 8% of patients. 12 Of note, BiTE therapy can also be associated with cardiovascular adverse events. For example with blinatumomab, CRS was reported for 12% of the patients and the most common cardiovascular adverse events included hypertension (5-8%), hypotension (12-14%), and sinus tachycardia (5-6%). Serious events such as MI, atrial arrhythmias, congestive heart failure, and cardiac arrest are overall rare (<0.5%). 32 While the exact rates of MACE either with CAR T-cells or BiTEs may vary, studies across multiple large centers, trials, and registries show that cardiovascular adverse events are not uncommon and it is important for both cardiologists and oncologists to understand this entity.

Surveillance and Management of Adverse Cardiac Effects
Currently, there are no established guidelines regarding risk stratification of CAR T-cell recipients for early detection and treatment of cardiovascular toxicities. Based on available data we propose careful pre-treatment screening of cardiovascular comorbidities using a baseline ECG, chemistry panel, troponin, B-Type Natriuretic Peptide (BNP), and transthoracic echocardiogram with global longitudinal strain (GLS). High-risk patients could include those greater than 60 years of age, those with elevated baseline troponin or BNP, those with demonstrated left ventricular dysfunction (baseline ejection fraction <55% or baseline abnormal GLS), or those with two or more high-risk co-morbidities. These include hypertension, hyperlipidemia, diabetes mellitus, chronic kidney disease, heart failure, history of arrhythmia, moderate to severe coronary artery disease, history of cardiotoxic chemotherapy such as anthracyclines, or history of previous chest radiation. 33 Patients identified as high-risk individuals for cardiovascular toxicities based on the above criteria should be referred to a cardio-oncologist for cardiac status evaluation prior to CAR T-cell treatment. While time to optimize medications may be limited due to the often aggressive nature of the malignancy requiring CAR T-cell therapy, a thorough risk assessment by a cardio-oncologist may help develop personalized clinical management strategies in anticipation of a possible cardiovascular adverse event for a higher-risk patient. We suggest that this includes a baseline ECG, baseline echocardiogram with strain, monitoring with telemetry if a history of arrhythmias, checking serum biomarkers such as troponin or BNP at days 0, 3, and 7 post CAR T-cell infusion and having a low threshold to repeat an echocardiogram with strain for assessment of early myocardial dysfunction if new heart failure symptoms, elevation in biomarkers, and/or high-grade CRS. Patients who develop high-grade CRS ( ≥ grade 2) after CAR T-cell infusion should be considered highrisk for cardiac toxicities such as new or worsening heart failure or new or worsening arrhythmias. These patients should be monitored closely with additional cardiac biomarkers, ECG, or echocardiography. Ultimately these patients should also follow up with a cardio-oncologist after any adverse event for close surveillance and further management.
Alongside corticosteroids and/or tocilizumab to treat CRS, in patients with evidence of cardiotoxicity GDMT or antiarrhythmic therapy should be utilized when clinically indicated. Currently, IL-6 inhibition with tocilizumab is the first-line treatment for severe CRS. Corticosteroids are primarily reserved for refractory CRS and are rapidly tapered after the CRS improves. This is primarily due to concerns about potential negative effects of corticosteroids on CAR T-cell efficacy though recent data suggest that high-dose dexamethasone and methylprednisolone may not have an impact on CAR T-cell efficacy or endurance. 34 Nonetheless, not much research has been done regarding the ability of prophylactic GDMT, corticosteroids, or tocilizumab to affect CAR T-cell-induced cardiac toxicities.
Similar to how patients undergo a perioperative risk assessment to facilitate informed decision-making, it will be useful to develop risk assessment tools for cardiac adverse events after CAR T-cell therapy. Currently, patients planned for noncardiac surgeries undergo stratification to provide a risk versus benefit calculation of surgical intervention weighed against the possibility of cardiac morbidity and mortality. 35 One commonly used risk calculator includes the revised cardiac risk index (RCRI) and the score helps delineate the percentage risk of major cardiac complications based on a variety of factors including kidney disease, diabetes requiring insulin use, history of ischemic heart disease, history of congestive heart failure, and others. 36 Though this certainly varies in the context of emergency, urgent, and elective surgeries, it provides a helpful framework for both clinicians and patients. 37 Currently there is insufficient data to construct such a calculator for CAR T-cell-associated cardiac toxicity, but in the future, it will be helpful to consistently collect additional data points so a stepwise algorithm may be developed. Since CAR T-cell therapy is often undertaken in urgent to an emergent situation, a certain degree of cardiac toxicity will likely have to be tolerated but as indications and uses for cellular therapy expand, a more precise periprocedure risk calculator will help inform awareness, earlier detection, and management, especially for high risk patients. It is necessary for clinical trial design to incorporate a systematic collection of data including details regarding medical comorbidities and preexisting cardiovascular risk factors and also data regarding cardiac function including baseline and interval ECGs, echocardiograms, and biomarker measurements.

Future Research Directions of CAR T-Cell Therapy
A key research area in CAR T-cell treatment cardiotoxicity is to understand which clinical variables are associated with post-treatment cardiotoxicity. Biomarkers, cardiac imaging, study design, and translational research are all critical areas for research needs (Figure 2).

Biomarkers
Future studies are needed to define the role of cardiac biomarker monitoring in patients undergoing CAR T-cell treatment. While other studies have examined biomarkers predictive of severe CRS including interferon-gamma, IL6, sgp130, and sIL6R, few studies exist with regard to biomarkers predictive of CAR T-cell cardiotoxicity. [38][39][40] Hu et al conducted a prospective study of 40 patients receiving CAR T-cell treatment and measured high-sensitivity troponin T (hsTropT) and N-terminal pro-B natriuretic peptide (NTproBNP) at baseline, day 1, day 7, and day 21 after CAR T-cell treatment with the hypothesis that an elevation of troponin could indicate damage to myocardium similar to its utility in ischemic cardiomyopathy and that an elevation of NTproBNP could be a surrogate for cardiac strain similar to its use in heart failure. Highly sensitive TropT did not vary significantly over the course of treatment, but NTproBNP was progressively elevated compared to baseline on days 1 and 7. 41 However, this study was limited by sample size and was not powered to assess the correlation between NTproBNP and likelihood or timeframe of developing a MACE. Other retrospective studies have noted a role for elevation in serum troponin in identifying patients at high risk for cardiac events post CAR T-cell infusion. [42][43][44] In particular, Alvi et al noted that of patients who had both pre-and post-CAR T-cell treatment troponin measurements, more than half of the patients had an elevation in serum troponin post CAR T-cell treatment (29/54, 54%) and 95% of high grade CRS (>2) events were preceded by an elevated troponin. 28 Furthermore, very little is known about the value of inflammatory biomarkers such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), ferritin, and other inflammatory cytokines such as interleukins (IL) or matrix metalloproteinases (MMP) in association with MACE. These markers are often measured in conjunction with assessing CRS but the overall timing and pattern of their rise and fall has not been well characterized in the context of cardiac toxicity. 38,45 As surrogates of systemic inflammation, they may be useful to trend during the treatment course. Ultimately more systematic research with larger sample sizes and pre-defined time points is needed to fully characterize their utility in relation to cardiotoxic adverse events.

Cardiac Imaging
Another prospective area of research is the utility of various imaging modalities in assessing CAR T-cell toxicities. To date, few studies have systematically employed serial echocardiograms to assess cardiac function. Ganatra et al reported new or worsening cardiomyopathy in 10% of CAR T-cell recipients but in this study serial echocardiograms were primarily done for patients experiencing higher-grade CRS or at the discretion of the treating physician. This may underestimate the true incidence of reduced ejection fraction and a prospective study with long-term, serial echocardiography would be informative for the future. 46 Although changes in left ventricular ejection fraction are the primary parameter to detect cardiomyopathy, novel echocardiographic techniques using speckle-tracking strain are also emerging as a tool for screening and monitoring cardiotoxic therapies. Subclinical left ventricular dysfunction can be detected by GLS measurements and can be a useful additive to ejection fraction and other echocardiographic parameters. 47,48 Shalabi et al showed that among 14 patients with strain measurements at baseline and during CRS, 9 patients had a relative reduction in GLS > 15%. 49 However, this is a small sample size among pediatric patients and additional studies are warranted. Cardiac magnetic resonance imaging (MRI) has also yet to be studied in the context of CAR T-cell cardiotoxicity. Cardiac MRI can often provide additional diagnostic data over echocardiograms and is useful to rule-out myocarditis but may be difficult to obtain quickly, especially in the acute toxicity period.

Study Design
Furthermore, longitudinal studies of the cardiotoxic effects should also be undertaken. Data so far suggests that decreased left ventricular ejection fraction related to cardiotoxicity in many cases may recover but very little systematic longitudinal monitoring exists for these cases. For example, one study of pediatric patients showed that persistent dysfunction at 6-month follow-up was rare, but this was limited by a sample size of 10 patients that had documented echocardiographic dysfunction among a total study population of 98 patients. 29 Additional serial longitudinal GLS measurements may also be a more sensitive way to detect subtle, persistent abnormalities. Ultimately despite the apparent recovery, questions of whether these CAR T-cell-associated cardiotoxic events may increase risk for future cardiac events given their long-term engraftment remain unanswered. 46 Given the limited follow-up and relatively small sample sizes of single-center studies it is crucial that cardiovascular toxicities be assessed in larger, prospective studies.
Special populations, such as pediatric and young adults receiving CAR T-cell treatment may have unique long-term cardiac sequelae and require monitoring. 49 Fitzgerald et al studied 39 pediatric and young adult patients undergoing CAR T-cell treatment for relapsed/refractory ALL. This study identified grade 3-4 CRS in 18 subjects and found that 10 suffered vasogenic shock requiring a vasoactive agent which took a median of 4 days to resolve. 50 Another study by Burstein et al showed that 24 of 98 patients experienced hypotension requiring inotropic support and 10 patients showed worsened echocardiographic systolic function. Factors associated with hypotension requiring inotropic support included higher pretreatment blast percentage on bone marrow biopsy, and baseline lower ejection fraction or diastolic dysfunction. 29

Translational Research
Several studies have focused on the correlation between CRS and cardiotoxicity. Emerging research suggests that vascular endothelial activation can contribute to the development of CRS and ICANS. 51,52 Therefore, blockade of tumor necrosis factor α (TNFα) and IL-1β may have therapeutic potential. 51 However, the role of endothelial activation, TNFα, and IL-1β in CAR T-cell-associated cardiac events has yet not been explored. CRS has also been hypothesized to play a role in CAR T-cell cardiotoxicity. Perhaps the acute onset, widespread immune and inflammatory activation precipitate a critical illness or stress-induced cardiomyopathy though more research in this area is needed. Research does suggest that early treatment with tocilizumab (anti-IL6 receptor antagonist) may help mitigate cardiotoxicity and research by Alvi et al showed that cardiovascular event risk increased 1.7-fold with each 12-hour delay to tocilizumab. 28 Direct cardiac injury is also another hypothesis. A case report of melanoma-associated antigen 3 (MAGE-A3) CAR T-cells showed cardiac histopathologic analysis with evidence of cardiac myonecrosis and T-cell infiltration in two patients who died from cardiogenic shock. 53 The cardiac toxicity in this case represented a cross-reactivity of the T-cell receptor to an unintended target antigen, titin, a sarcomeric protein expressed in cardiomyocytes. Another example of on-target, off-tumor toxicity includes possible cross-reactivity between anti-mesothelin CAR T-cells with normal mesothelin expressed on serous cells of the pleura and pericardium causing pleuropericarditis, though this was not shown in a recent phase I trial. 54

Insights for Developmental Cardiovascular Immunotherapies
While CAR T-cell therapy may be associated with cardiovascular toxicities, recent research has also shown success in utilizing CAR T-cell therapy to attenuate cardiac fibrosis in murine models. 24 While previous CAR T-cells targeting fibroblast activation protein (FAP) were used in a mouse model of hypertensive cardiac injury, 55 Rurik et al developed a unique method to generate transient CAR T-cells in vivo to bypass the need for ex vivo cell manufacturing and to minimize long-term side effects of persistently inhibiting the anti-fibrotic response. In this study targeted lipid nanoparticles were able to reprogram T-cells and analysis of a murine model of heart disease showed that the approach was able to reduce fibrosis and restore cardiac function. 24 Other developing cardiovascular immune therapies include engineered CAR Treg cells directed against atherosclerosisrelevant epitopes to suppress local inflammation and developing tolerogenic Treg-based vaccines to prevent and treat cardiovascular diseases. 56 Overall this highlights how an improved understanding of T-cell biology in cardiovascular disease can be leveraged for both better management of cardiovascular adverse events and to expand CAR T-cell use for directed immunotherapy of cardiovascular diseases.

Conclusions
A better understanding of patient characteristics before and after undergoing CAR T-cell therapy will help inform improved therapy selection, prognosis, and management of patients receiving cellular therapies. There are several areas of future research needs, including biomarkers, cardiac imaging, longitudinal studies, special attention to sub-populations and translational research on mechanisms of cardiotoxicity. Increasing knowledge of the adaptive immune response and a better understanding of both engineered and non-engineered T-cell function in cardiovascular tissues can help inform future cardiovascular immunotherapies. Furthermore, as cellular therapies move beyond T-cells and expand into chimeric antigen NK cells and macrophages, it is important to keep in mind potential cardiotoxicities and incorporate cardiac parameters prospectively into the design of clinical trials. This includes routine data collection about cardiac function at prespecificed intervals by assessing laboratory and imaging parameters through BNP, troponin, electrocardiography, and echocardiography with GLS. While this may not mitigate the risk of MACE entirely, it would help better understand the pathophysiology and evolution of cardiac injury after cellular therapies and could improve strategies for early detection and management.


Roles of Endomembrane Alkali Cation/Proton Exchangers in Synaptic Function and Neurodevelopmental Disorders

Endomembrane alkali cation (Na+, K+)/proton (H+) exchangers (eNHEs) are increasingly associated with neurological disorders. These eNHEs play integral roles in regulating the luminal pH, processing, and trafficking of cargo along the secretory (Golgi and post-Golgi vesicles) and endocytic (early, recycling, and late endosomes) pathways, essential regulatory processes vital for neuronal development and plasticity. Given the complex morphology and compartmentalization of multipolar neurons, the contribution of eNHEs in maintaining optimal pH homeostasis and cargo trafficking is especially significant during periods of structural and functional development and remodeling. While the importance of eNHEs has been demonstrated in a variety of non-neuronal cell types, their involvement in neuronal function is less well understood. In this review, we will discuss their emerging roles in excitatory synaptic function, particularly as it pertains to cellular learning and remodeling. We will also explore their connections to neurodevelopmental conditions, including intellectual disability, autism, and attention deficit hyperactivity disorders.

INTRODUCTION
In neurons, accurate execution of processes along the secretory and endolysosomal pathways is essential for the normal development, maturation, and remodeling of synaptic connections. For instance, glycosylation of newly synthesized neurotransmitter transporters (Melikian et al., 1996;Martinez-Maza et al., 2001;Li et al., 2004;Cai et al., 2005), neurotransmitter receptors (Jiang et al., 2006;Kaniakova et al., 2016;Sinitskiy et al., 2017), and voltage-gated ion channels (Zona et al., 1990;Ednie and Bennett, 2012;Baycin-Hizal et al., 2014;Scott and Panin, 2014) enhances their stability, membrane trafficking and/or activities that are crucial for membrane excitability, neurotransmission, and ultimately learning and memory (Matthies et al., 1999;Inaba et al., 2016). Likewise, optimal performance of the endolysosomal system is vital for neurotrophin signaling, presynaptic vesicle recycling, axonal growth cone migration, and synaptic plasticity (Morgan et al., 2013;Bowen et al., 2017;Terenzio et al., 2017;Hiester et al., 2018;Kiral et al., 2018;Naslavsky and Caplan, 2018). Unlike other cells in the body, the morphologies of neurons are quite elaborate, which creates additional complexity in the compartmentalization and regulation of protein trafficking between their somata, dendrites, and axons. In recent years, there has been increasing awareness that disruptions in biosynthetic and endolysosomal functions underlie several neurodevelopmental and neurodegenerative disorders involving synaptic deficits, eventually leading to cognitive impairments (Lefebvre et al., 2003;Aronica et al., 2005;Nixon, 2005;Baloyannis, 2014;Maxfield, 2014;Schreij et al., 2016;Wang et al., 2016;Bingol, 2018;Winckler et al., 2018). The underlying factors are assuredly multifactorial and complex. While significant advances have been made in identifying many of these factors, a detailed appreciation of how they cause neuronal dysfunction has yet to be fully realized. Increasing evidence has revealed a central role for pH homeostasis of endomembrane compartments in central nervous system function and neurological disorders (Jentsch and Pusch, 2018;Sou et al., 2019;Collins and Forgac, 2020;Khosrowabadi and Kellokumpu, 2020;Prasad and Rao, 2020). For the purposes of this review, we will highlight the importance of the endosomal system to excitatory synaptic plasticity in the brain, and then focus our discussion on the significant contributions of alkali cation (Na + or K + )/proton (H + ) exchangers (NHEs) (also referred to as the SLC9 gene family) to this process.

ENDOSOMAL TRAFFICKING IN NEURONAL PLASTICITY
Studies investigating cognitive deficits commonly focus on the hippocampus, a medial temporal lobe structure that has long been implicated in learning and memory (Scoville and Milner, 1957;Penfield and Milner, 1958;Stuchlik, 2014). Pyramidal neurons in the hippocampus are dotted by dendritic spines, small (1 µm) heterogenous protrusions that emanate from the dendritic shaft and serve as the sites of excitatory postsynaptic connections (Gray, 1959;Bourne and Harris, 2008;McKinney, 2010). In general, dendritic spines contain a "head" structure joined to the dendritic shaft by a thinner "neck" apparatus (Harris et al., 1992). Under electron microscopy, the heads of dendritic spines contain an electron-dense region known as the postsynaptic density (PSD), which lies in direct apposition to the presynaptic active zone (Lei et al., 2016;Borovac et al., 2018) ( Figure 1). The PSD typically contains hundreds of proteins involved in excitatory postsynaptic signaling, including ionotropic glutamatergic α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptors (AMPARs) and N-methyl-Daspartate receptors (NMDARs). AMPARs are non-specific cation channels that primarily conduct Na + and K + ions and are responsible for propagating fast excitatory neurotransmission. NMDARs are also permeable to monovalent cations but also conduct divalent ions such as Ca 2+ which can activate downstream signaling cascades that alter synaptic strength (McKinney, 2010). As larger spines typically contain a larger PSD and more AMPARs, it is widely believed that spine size

ENDOMEMBRANE PH HOMEOSTASIS
The internal pH of exocytic and endocytic compartments is an important determinant of their function and dynamics (Casey et al., 2010). For instance, the pH milieu of the endoplasmic reticulum (ER) is near neutral (pH~7.2) but becomes increasing acidic from the cis-Golgi network (pH~6.7) through to the trans-Golgi network (pH~6.0) ( Figure 2). Secretory vesicles can attain even higher H + concentrations (pH~5.2). Acidification of compartments along the biosynthetic pathway is important for proper post-translational processing, sorting and transport of newly synthesized proteins and lipids. Likewise, endocytosed membrane-bound proteins from the cell surface that are targeted for lysosomal degradation are subjected to a gradient of acidification from early endosomes (EE, pH~6.3) to late endosomes (LE, pH~5.5) and, finally, lysosomes (pH~4.7). Conversely, internalized cell-surface proteins destined for transport back to the plasma membrane may be trafficked through more alkaline recycling endosomes (RE, pH~6.5). Endolysosomal pH is involved in a number of functions, including 1) cargo sorting, 2) receptor-ligand dissociation and processing, 3) the maturation and transport of endolysosomal vesicles, 4) membrane protein and receptor recycling, and 5) the activity of luminal enzymes including degradative hydrolases (Casey et al., 2010;Hu et al., 2015). The proper regulation of luminal pH is thus vital for establishing the identity and function of each compartment. Vesicular acidification in both the secretory and endolysosomal pathways is primarily achieved by the vacuolar H + -ATPase (V-ATPase), an evolutionarily conserved multisubunit complex that utilizes the chemical energy present in ATP to pump H + from the cytosol into the endomembrane lumen (Casey et al., 2010;Collins and Forgac, 2020;Vasanthakumar and Rubinstein, 2020). Because V-ATPase-mediated activity transports only H + independently of other ions, this results in the generation of an inside-positive voltage gradient across organellar membranes that gradually impedes additional H + influx, limiting acidification. However, this effect is offset by counterion conductances (anion influx or cation efflux). The most prominent counterion conductance is mediated by members of the CLC family of voltage-gated chloride channels and transporters, specifically CLC3-7, which localize to secretory and endolysosomal compartments and facilitate the entry of negatively-charged Cl − anions to neutralize the membrane potential and allow for progressive acidification (Gentzsch et al., 2003;Guzman et al., 2015;Jentsch and Pusch, 2018). Notably, these CLC family members act as electrogenic 2Cl − /1H + exchangers and can thus serve as both a mechanism of compensatory anion influx as well as H + efflux (Picollo and Pusch, 2005;Scheel et al., 2005;Guzman et al., 2013;Rohrbough et al., 2018). However, CLCs are not the sole source of H + efflux, as a significant fraction of this H + leakage is also attributed to endomembrane members of the (Na + or K + )/H + exchanger (NHE) gene family (Nakamura et al., 2005;Orlowski and Grinstein, 2007).

THE NHE FAMILY
NHEs are a family of evolutionarily conserved secondary active transporters that couple the countertransport of monovalent alkali cations (such as Na + , K + , or Li + ) for H + across biological membranes. To date, 13 distinct mammalian genes from three phylogenetically distinct categories have been identified, including NHE1 through NHE9 (encoded by the SLC9A1-SLC9A9 genes), Na + /H + antiporters 1 and 2 (NHA1-2, encoded by the SLC9B1-2 genes), and the recently characterized NHE10 and NHE11 (SLC9C1 and SLC9C2 genes, respectively) (Brett et al., 2005a;Orlowski and Grinstein, 2011;Fuster and Alexander, 2014;Pedersen and Counillon, 2019). NHE proteins differ in their sequence length, drug sensitivity, cation selectivity, and tissue and subcellular localization, with some being predominantly localized to specific subdomains of the cell surface and others to intracellular organelles. Amongst the SLC9A isoforms, five (NHE1 through NHE5) are primarily active at the plasma membrane, while the remaining four (NHE6 through NHE9) localize mainly in intracellular compartments. Plasmalemmal NHEs mediate the electroneutral exchange of extracellular Na + (or Li + ) for cytosolic H + (stoichiometry of 1:1 or 2:2) (Aronson, 1985;Fuster et al., 2008) and are mainly responsible for regulating systemic and cytosolic pH. In contrast, endomembrane NHEs mediate the electroneutral transport of luminal H + for a cytosolic cation (such as Na + or K + , with the latter being more prominent given its higher intracellular concentration) to alkalinize organellar pH (Numata and Orlowski, 2001;Brett et al., 2005b;Nakamura et al., 2005). Together, the NHE/SLC9A transporters regulate intracellular and organellar pH, volume, and osmolality, which in turn influences vesicular trafficking, signaling, cell growth and migration. The functions of the SLC9B and SLC9C isoforms are less well characterized. SLC9B isoforms are broadly expressed and have been implicated in hypertension (Xiang et al., 2007;Chintapalli et al., 2015;Kondapalli et al., 2017), insulin secretion (Deisl et al., 2013;Deisl et al., 2016) and sperm motility and fertility (Chen et al., 2016). By contrast, expression of the SLC9C isoforms is largely confined to testis where they are also essential for sperm function (Wang et al., 2003;Quill et al., 2006;Wang et al., 2007;Windler et al., 2018;Cavarocchi et al., 2021).
While NHEs are typically categorized as acting at the plasma membrane or endomembrane compartments, many of them shuttle continuously between cellular locations in a cell-or stimulus-dependent manner. Most notably, upon exit from the ER, the endomembrane NHEs (eNHEs) are trafficked through the secretory pathway and transiently appear at the cell surface prior to being internalized to their respective compartments (Orlowski and Grinstein, 2007). Moreover, although the phylogenetically-related epithelial-enriched NHE3 and neuralenriched NHE5 isoforms are often categorized as cell surface NHEs, they are also found within a pool of intracellular vesicles that rapidly shuttle to and from the plasma membrane in response to diverse stimuli (D'Souza et al., 1998;Janecki et al., 1998;Kurashima et al., 1998;Szászi et al., 2002;Yang et al., 2002;Donowitz et al., 2009;Bobulescu et al., 2010;Diering et al., 2011;Lukashova et al., 2011;Lukashova et al., 2013;Jinadasa et al., 2014). Indeed, the tissue expression and spatiotemporal membrane trafficking of these NHEs is a significant determinant of their function. For instance, NHE3 is present at the apical surface as well as intracellular vesicles of renal and gastrointestinal epithelia and plays critical roles in regulating not only ion secretion and resorption, indirectly mediating the transmembrane movement of fluid, but also regulating the pH of newly formed endosomes and internalization of select cargo (D'Souza et al., 1998;Gekle et al., 2001;Gekle et al., 2004;Orlowski and Grinstein, 2011;Fuster and Alexander, 2014;Pedersen and Counillon, 2019). Similarly, NHE5 has been detected in endosomes of C6 glioma cells that partially overlap markers for recycling endosomes where it has been implicated in modulating endosomal pH as well as cell surface delivery and signalling of the hepatocyte growth factor receptor (HGFR; also called the MET receptor tyrosine kinase) and epidermal growth factor receptor (Fan et al., 2016;Kurata et al., 2019). By contrast, other isoforms such as NHE6, NHE7, NHE8, and NHE9 are widely expressed where they reside mainly in intracellular compartments and, to a limited extent, at the plasma membrane (Nakamura et al., 2005;Orlowski and Grinstein, 2007;Ohgaki et al., 2011). Though these isoforms primarily regulate endomembrane pH homeostasis and trafficking, they can also be active at the cell surface of specialized cells (Goyal et al., 2005;Hill et al., 2006;Zhang et al., 2007;Xu et al., 2008).

ROLES OF NHES IN THE CENTRAL NERVOUS SYSTEM
Increasing attention has recently been paid to the function of NHEs in brain. Earlier experiments investigating NHEs in excitatory synaptic plasticity showed that NHE blockade using the broad-spectrum inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) was found to significantly improve LTP maintenance in rat hippocampal slices (Ronicke et al., 2009). While the specific NHE isoform(s) responsible for this phenomenon was not determined, it is evident that NHE-mediated pH modulation can play a critical role in synaptic transmission and remodeling. Accordingly, disruption of some of these isoforms has been associated with neuropathologic conditions. Ablation of NHE1 in mice leads to a severe neurodegenerative phenotype associated with ataxia, seizures, and postnatal lethality (Cox et al., 1997;Bell et al., 1999). NHE1 has since been shown to regulate the release of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA) (Jang et al., 2006;Dietrich and Morad, 2010;Bocker et al., 2019) and may thus play a larger role in mediating cellular excitability. Likewise, mutations in human NHE1 cause Lichtenstein-Knorr syndrome, an autosomal recessive disorder characterized by hearing loss and cerebellar ataxia (Guissart et al., 2015). Disruption of human NHE1 function has also been associated with other forms of ataxia, spastic paraplegia, intellectual disability, and epilepsy (Zhu et al., 2015;Iwama et al., 2018;Mendoza-Ferreira et al., 2018). Moreover, genetic variants in the human NHE7 have recently been shown to cause a non-syndromic form of intellectual disability accompanied by macrocephaly, minimal speech, muscular weakness and hypotonia (Khayat et al., 2019). NHE7 resides within the Golgi complex, accumulating predominantly in the trans-Golgi network and post-Golgi vesicles (Numata and Orlowski, 2001;Lin et al., 2005) where it regulates luminal pH as well as glycosylation of exported cargos (Khayat et al., 2019). Although further insights into the neuronal function of NHE7 are presently lacking, these findings illuminate the role of organellar pH in regulating learning and cognition in the brain.
The roles of two other closely-related (58% amino acid identity) eNHEs, NHE6, and NHE9, have also received considerable attention. Both eNHEs are expressed ubiquitously, though NHE6 is particularly enriched in brain (see https://www.ncbi.nlm.nih.gov/gene/10479 for SLC9A6 and https://www.ncbi.nlm.nih.gov/gene/285195 for SLC9A9). These eNHEs localize predominantly to discrete but overlapping pools of EEs and REs (Nakamura et al., 2005;Orlowski and Grinstein, 2007;Deane et al., 2013) (Figure 1). In the human genome, the SLC9A6/NHE6 and SLC9A9/NHE9 genes localize at chromosomal positions Xq26.3 and 3q24, respectively. In humans, several mRNA splice-variants of NHE6 have been documented in research databases (https://www.ncbi.nlm.nih. gov/gene/10479). Thus far, only two of these, NHE6v1 and NHE6v2 (originally called NHE6.1 and NHE6.0, respectively) have been examined at the protein level, with the former containing an additional 32 amino acid insertion within its second extracellular loop. This insert contains an additional glycosylation site, though its functional significance, if any, remains uncertain. As expected, overexpression of NHE6 (Ohgaki et al., 2010;Xinhan et al., 2011;Ilie et al., 2016) and NHE9 (Kondapalli et al., 2013) in cultured cells resulted in the elevation of endosomal pH, whereas knock-down or ablation had the opposite effect (Ohgaki et al., 2010;Xinhan et al., 2011;Ouyang et al., 2013;Ullman et al., 2018). Most notably, deleterious mutations in these eNHEs have been directly associated with neurodevelopmental disorders. Mutations in NHE6 result in Christianson syndrome (CS), a monogenic disorder causing severe X-linked intellectual disability (XLID), non-verbalism, epilepsy, truncal ataxia, sleep disturbances, postnatal microcephaly, and autistic traits (Christianson et al., 1999;Gilfillan et al., 2008;Tarpey et al., 2009;Garbern et al., 2010;Schroer et al., 2010;Mignot et al., 2013;Ilie et al., 2019;Gruber et al., 2022). While CS is considered to be a rare disorder, SLC9A6 is one of the six most commonly mutated loci in patients with XLID (Pescosolido et al., 2014), suggesting that mutations in this gene may be more common than previously thought. By contrast, variants in SLC9A9/NHE9 are associated with attention deficit hyperactive disorder (ADHD) (de Silva et al., 2003;Lasky-Su et al. , 2008) and autism spectrum disorder with epilepsy (ASD) (Morrow et al., 2008). Curiously, findings from post-mortem tissue of individuals diagnosed with idiopathic ASD have suggested a downregulation of NHE6 expression with a concomitant increase in that of NHE9 (Schwede et al., 2013), suggesting a possible reciprocity in the function of these two eNHEs in the development of ASD. Indeed, genes encoding endosomal regulators are highly dominant among risk genes for ASD, illustrating the importance of this system and, by extension, these eNHEs in the function of neurons and synapses (Patak et al., 2016). The mechanisms by which genetic disruptions in eNHEs result in such severe disorders of the nervous system are poorly understood and remain to be elucidated.

ROLES OF NHES IN VESICULAR TRAFFICKING
Given their subcellular locations, it is perhaps not surprising that these eNHEs would play critical roles in endocytic cargo trafficking. Even in non-neuronal cells, one of the first physiological roles identified for NHE6 function was in the Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 892196 5 development of cellular polarity. In liver hepatoma HepG2 cells, which possess distinct apical and basolateral surface domains, knock-down of NHE6 selectively decreased endosomal pH and disrupted transcytotic recycling of proteins to the apical domain, thereby disrupting the development of proper cellular polarity (Ohgaki et al., 2010). NHE6 is further implicated in clathrin/ adaptor protein 2 (AP2)-mediated endocytosis (CME) of cellsurface proteins. In HeLa cells, NHE6 colocalizes strongly with both clathrin and transferrin, a cellular iron carrier that is classically used to study CME into recycling endosomes (Xinhan et al., 2011;Ilie et al., 2016). Accordingly, knockdown of NHE6 attenuated transferrin uptake in HeLa cells, which was indicative of a role for NHE6 in trafficking of recycling endosomal cargo. However, manipulation of NHE6 expression did not impair CME of the activated epidermal growth factor, suggesting that NHE6 regulates only a subset of cargo internalized by CME (Xinhan et al., 2011;Ilie et al., 2016). NHE9 similarly colocalizes with transferrin in mouse primary cortical astrocytes, in which overexpressing NHE9 significantly enhanced transferrin uptake (Kondapalli et al., 2013). Interestingly, siRNA knockdown of NHE9 did not alter transferrin uptake, suggesting some degree of compensation from NHE6 in the absence of NHE9. Parenthetically, a recent report showed that NHE9 is present within microvascular endothelial cells that line the blood-brain barrier and mediates iron delivery to the brain (Beydoun et al., 2017). In response to iron starvation, NHE9 levels are upregulated, alkalinizing endosomal pH and promoting the recycling of transferrin receptors back to the cell surface to facilitate additional iron uptake (Beydoun et al., 2017). The importance of NHE6 and NHE9 in regulating transferrin uptake and CME in general may thus be context-and cell-type specific. It should be noted that in neurons, AMPARs, NMDARs, ionotropic GABA A receptors, and other cell-surface proteins are predominantly internalized via CME (Kittler et al., 2000;Hanley, 2018). Therefore, alterations in eNHE function are predicted to perturb CME mechanisms in neurons and disrupt the regulation of neurotransmission and remodeling of both excitatory and inhibitory synapses.

ROLES OF ENDOSOMAL NHES IN NEURODEVELOPMENT
Both NHE6 and NHE9 are critical during neurodevelopment. During murine embryogenesis, NHE6 is strongly present in developing fiber tracts across multiple brain areas, including the cortex, striatum, thalamus and hippocampus (Deane et al., 2013;Ouyang et al., 2013). NHE6 expression is then persistent throughout the postnatal brain, although it is significantly upregulated at postnatal day (PD) 50 during a period of intense synaptic pruning and refinement (Deane et al., 2013). In contrast, NHE9 expression is relatively low during development and begins to increase only postnatally, peaking at around PD 50 and declining thereafter (Ullman et al., 2018). Adult brain NHE9 expression appears to be restricted to the olfactory bulb, superficial cortical layers, and hippocampus (Lein et al., 2007). At the cellular level, NHE6 and NHE9 are broadly present within EEs and REs throughout the axons, somata, and spines of hippocampal pyramidal neurons (Deane et al., 2013;Ouyang et al., 2013;Ullman et al., 2018). These observations confirm the involvement of eNHEs in neuronal endosomes and suggest that they may function in long-range trafficking throughout neuronal compartments as well. Given that NHE6 is a critical mediator in the development of cellular polarity (Ohgaki et al., 2010), it is unsurprising that neurons deficient in functional NHE6 display reductions in axodendritic branching (Ouyang et al., 2013;Ilie et al., 2014;Ilie et al., 2016;Gao et al., 2019;Lizarraga et al., 2021). Accordingly, male Nhe6 knock-out (KO) mice exhibit a pronounced overall undergrowth of the brain in addition to progressive volumetric losses in the cortex, striatum, hippocampus and cerebellum throughout adulthood (Xu et al., 2017). These reductions in neuronal branching and brain size are in accordance with the postnatal microcephaly commonly observed in human CS patients (Christianson et al., 1999;Pescosolido et al., 2014), which is highly suggestive of a reduction in adolescent brain development when NHE6 function is reduced or ablated. This has been hypothesized to arise from reduced activity of brain-derived neurotrophic factor (BDNF) (Ouyang et al., 2013), a critical neurotrophin that activates intracellular pathways regulating neuronal outgrowth, differentiation, plasticity and survival (Huang and Reichardt, 2001;Chao, 2003). Typically, binding of BDNF to its high affinity receptor tyrosine receptor kinase B (TrkB) results in the dimerization, autophosphorylation and endocytosis of the phosphorylated TrkB complex into signaling endosomes (SEs) (Xu et al., 2000;Rajagopal et al., 2004;Rex et al., 2007;Koleske, 2013). These SEs are then capable of activating downstream signaling cascades locally near the site of endocytosis to regulate neurite branching and elaboration, or undergo longrange retrograde transport back to the cell soma to activate transcriptional programs in the nucleus (Harrington and Ginty, 2013;Cosker and Segal, 2014). Importantly, this sorting decision can be influenced by the internal acidity of these SEs (Harrington et al., 2011), again underscoring the critical role of proper endosomal pH regulation. In hippocampal neurons, NHE6 colocalizes strongly with TrkB, suggesting that NHE6 is indeed involved in regulating the luminal pH of TrkB-containing SEs. Moreover, phosphorylated TrkB levels are reduced in Nhe6 KO neurons (Ouyang et al., 2013) (Figure 3). These results engendered the hypothesis that the overacidification of endosomal pH induced by a lack of NHE6 function may lead to the mistargeting of TrkB to more acidic lysosomes to be degraded by resident hydrolases. Indeed, application of exogenous BDNF was sufficient to restore phosphorylated TrkB levels and neurite branching in cultured hippocampal KO neurons (Ouyang et al., 2013), implying that reversing the attenuation in BDNF/TrkB signaling was the root cause of these morphological impairments in neurons grown in vitro. Given that BDNF/TrkB signaling can also influence synaptic function and plasticity (Leal et al., 2017), it is conceivable that excitatory neurotransmission may also be impacted in murine Nhe6 KO hippocampal neurons. Augmenting signaling through this pathway using specific TrkB agonists may thus prove to be beneficial in alleviating neurological deficits in CS patients Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 892196 6 harbouring SLC9A6 mutations, as hippocampal BDNF/TrkB signaling is likely to be similarly diminished in these individuals.
How does the loss of eNHE function impact animal behaviour? Homozygous male Nhe6 KO (Slc9a6 -/Y ) mice exhibit several neurobehavioural abnormalities that resemble clinical phenotypes seen in CS patients. Perhaps the most evident are deficits in locomotor control, as revealed by their relatively poor performance in rotarod and balance beam tasks when compared to age-and sex-matched wild-type (WT) mice (Stromme et al., 2011). These mice further exhibit hyperactivity and anxiety-type behaviours in open field paradigms (Stromme et al., 2011;Sikora et al., 2016), as well as impaired visuospatial learning (Sikora et al., 2016) and diminished sensitivity to pain and pressure stimuli (Kerner-Rossi et al., 2019;Petitjean et al., 2020). Interestingly, female heterozygous mice (Slc9a6 +/− ) show an intermediate phenotype compared to homozygous KO males (Slc9a6 -/Y ) and WT male and female mice (Sikora et al., 2016), mirroring clinical findings in female carriers of deleterious SLC9A6 alleles (Christianson et al., 1999;Pescosolido et al., 2019;Nan et al., 2022). These observations in heterozygotes are suggestive of a gene dosage effect of SLC9A6 mutations upon these phenotypes, as the extent (or skewing) of X-inactivation (Belmont, 1996;Plenge et al., 2002) of either the WT or mutant SLC9A6 allele in females likely accounts for the wide heterogeneity of their traits. Conversely, Slc9a9/Nhe9 KO mice do not show obvious changes in gross hippocampal morphology or impairments in locomotion, anxiety, smell, or pain sensitivity (Yang et al., 2016;Ullman et al., 2018). However, they do exhibit classic characteristics of ASD, including repetitive behaviours and impaired social and olfactory function (Yang et al., 2016;Ullman et al., 2018). These differences allude to an intriguing divergence in how the loss of either eNHE impacts brain function in mammals and reflects the differing phenotypes observed in human patients harbouring genetic alterations in either NHE6 or NHE9.

ROLES OF ENDOSOMAL NHES IN EXCITATORY SYNAPTIC FUNCTION
These behavioural findings clearly establish the importance of eNHE function in the formation, function and plasticity of excitatory synapses. The precise roles of these eNHEs within the tripartite synapse (i.e., presynaptic and postsynaptic sites as well as neighbouring astrocytes) are uncertain, but recent studies have implicated their involvement in both pre-and post-synaptic vesicle function (Figure 1). In the presynaptic terminal, H + import into synaptic vesicles (pH~5.7) is necessary for the loading of neurotransmitters (Miesenbock et al., 1998;Goh et al., 2011), underscoring the importance of vesicular pH regulation in this process. In the axons of cultured hippocampal neurons, signals of immunolabelled NHE6 and NHE9 endosomes overlap partially with synaptic vesicle 2 (SV2), a marker of presynaptic terminals (Ouyang et al., 2013;Ullman et al., 2018). NHE6 endosomes have also been observed in glutamatergic and GABAergic nerve terminals via mass spectrometry (Gronborg et al., 2010) as well as in purified glutamate-positive synaptic vesicles (Preobraschenski et al., 2014), where it may regulate vesicular cation transients during neurotransmitter loading. It is thus plausible that a reduction in NHE6 function could deleteriously impact quantal glutamate release. Indeed, fiber volleys recorded from area CA1 in Nhe6 KO hippocampi are significantly reduced in amplitude when compared to WT. As these recordings are reflective of presynaptic FIGURE 3 | Schematic of impaired endosomal pH homeostasis and AMPAR and TrkB trafficking leading to defective synaptic potentiation and behavioural learning in NHE6 KO versus WT mice. Potential treatment strategies (i.e., elevate endolysosomal pH, enhance TrkB activation) to address these deficiencies in KO are indicated. Thickness of arrows represents relative extent of AMPA transport (black) or TrkB receptor signaling (gray). EE, early endosome; RE, recycling endosome; LE, late endosome; Ly, lysosome; BDNF, brain-derived neurotrophic factor; PSD95, postsynaptic density protein-95; cargo X, undefined membrane cargo.
Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 892196 7 firing and activation properties, this finding implies a possible loss in the number of overall functional synapses (Ouyang et al., 2013). However, WT and Nhe6 KO do not significantly differ in the amplitude of paired-pulse ratio recordings at CA3-CA1 synapses (Ouyang et al., 2013), suggesting that the basal vesicular release properties of individual synapses may not be impacted by the loss of NHE6. Indeed, NHE6 has only been detected in a subset of synaptic boutons (Deane et al., 2013). Hence, a broad role for NHE6 in synaptic vesicle pH homeostasis and neurotransmitter uptake is unclear. Further investigations into the precise role of NHE6 within the presynaptic terminal are thus warranted to better understand the possible roles of this isoform in neurotransmitter loading and release.
By contrast, a recent study implicated NHE9 in presynaptic release. In contrast to studies in Nhe6 KO, fiber volley amplitudes recorded from hippocampal area CA1 appeared to be comparable between WT and Nhe9 KO, and thus the absence of NHE9 does not seemingly impact vesicular glutamate loading (Ullman et al., 2018). However, Nhe9 KO CA3-CA1 synapses showed a significant increase in paired-pulse ratio, suggestive of a decrease in presynaptic release probability. This supposition was further supported by measurements showing that exocytosis of synaptic vesicles was impaired in Nhe9 KO tissue due to a reduction in presynaptic Ca 2+ influx. This was attributed to overacidification of endosomes in the absence of NHE9, as transient vesicle alkalinization using the V-ATPase inhibitor bafilomycin was sufficient to restore presynaptic Ca 2+ entry in Nhe9 KO neurons. This suggests that proper endosomal pH regulation in the axonal bouton is indeed a critical component in the trafficking or function of proteins regulating Ca 2+ dynamics and neurotransmitter release. Further work into the involvement of NHE9 in regulating both neurotransmitter loading and presynaptic Ca 2+ homeostasis will be necessary to elucidate its precise roles in axon boutons.
To date, the roles of NHE6 are perhaps better understood in relation to postsynaptic function. NHE6 accumulates at the base and head regions of dendritic spines and colocalizes with the excitatory postsynaptic scaffolding molecule PSD95 (Deane et al., 2013;Ouyang et al., 2013) (Figure 1). Furthermore, NHE6 colocalizes strongly with the GluA1 subunit of the AMPAR and is rapidly recruited to spines in response to NMDARmediated chemical LTP (Deane et al., 2013). Presumably, NHE6-mediated trafficking of AMPAR-containing vesicles is necessary for excitatory synaptic function and plasticity. As such, Nhe6 KO CA1 pyramidal neurons demonstrated a marked reduction in overall dendritic spine density, with a loss of larger, more mature, dendritic spines concomitant with an increase in immature protrusions when compared to WT (Ouyang et al., 2013). These results strongly suggest that a complete loss of NHE6 function may reduce excitatory synaptic strength and activity-dependent remodeling, which has since been investigated with NHE6 mutants (discussed further below). In contrast, Nhe9 KO neurons display comparable axodendritic branching and dendritic spine density to WT cells (Ullman et al., 2018). Nonetheless, electrophysiological recordings from Nhe9 KO CA1 pyramidal neurons revealed cell-autonomous impairments of AMPAR and NMDAR function, suggesting that the loss of NHE9 can also alter glutamatergic neurotransmission in a pleiotropic manner (Ullman et al., 2018). Curiously, NHE9 does not appear to be involved in the trafficking of the AMPAR subunits, nor does the loss of NHE9 impact the surface expression of AMPARs or NMDARs (Ullman et al., 2018). Further insights into the role of NHE9 in mediating glutamatergic receptor trafficking and function are therefore necessary to better understand how changes in NHE9 function impact excitatory neurotransmission.
Both NHE6 and NHE9 are also expressed in astrocytes (Deane et al., 2013;Kondapalli et al., 2013;Ullman et al., 2018). With regards to NHE9, one report found that astrocytes lacking NHE9, or expressing ASD-associated variants in SLC9A9, exhibited a significant reduction in endosomal pH compared to those transfected with WT NHE9 (Kondapalli et al., 2013). Astrocytes expressing these ASD-associated SLC9A9 variants also failed to upregulate the surface expression of excitatory amino acid transporter 1 (EAAT1), resulting in a decrease in glutamate uptake when compared to overexpression of WT NHE9 (Kondapalli et al., 2013). Altered glutamate clearance may thus underlie the aforementioned disturbances in AMPAR and NMDAR function in Nhe9 KO neurons (Ullman et al., 2018), although direct assessments of excitatory transmission in cells expressing these NHE9 mutants have yet to be performed. Moreover, excess glutamate in the synaptic cleft likely imbalances the ratio of excitatory and inhibition transmission, which could account for the co-morbidity of epilepsy frequently observed in ASD (Kondapalli et al., 2014). Whether NHE6 also traffics EAAT1 in astrocytes is presently unclear, although magnetic resonance spectroscopy data of CS patients have shown an increase in glutamate concentration in the brain that may indicate enhanced excitatory transmission (Gilfillan et al., 2008;Schroer et al., 2010). Indeed, Nhe6 KO mice do exhibit a pronounced increase in activated astrocytes and microglia within their gray matter (Xu et al., 2017;Kerner-Rossi et al., 2019). This observation may be a response to the heightened neurodegeneration detected in CS patients (Christianson et al., 1999;Gilfillan et al., 2008;Garbern et al., 2010;Schroer et al., 2010) and NHE6 KO mice (Stromme et al., 2011;Xu et al., 2017), but it could also be indicative of possible neuroinflammation which may not directly arise from its role in astrocytes. Interestingly, though not covered in this review, diminished NHE6 expression has also been detected in astrocytes associated with Alzheimer's disease, suggesting a broader role for NHE6 is neurodegenerative pathophysiology (Prasad and Rao, 2018a;Prasad and Rao, 2018b). Taken together, it is evident that both NHE6 and NHE9 play very specific roles throughout the tripartite synapse, although important details remain to be discovered with regards to exactly which functions both eNHEs play within each compartment.
Tangentially, though not strictly considered an eNHE, the plasmalemmal NHE5 isoform also appears to play a role in postsynaptic remodeling and signaling. An intriguing study showed that in response to NMDAR-mediated chemical LTP, NHE5-containing vesicles are also recruited to, and undergo exocytosis at, dendritic spines. The addition of NHE5 at the postsynaptic membrane functions to extrude H + into the synaptic Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 892196 8 cleft to negatively regulate NMDAR activation and restrict excessive spine expansion (Diering et al., 2011). Rapid recruitment of NHE5 from intracellular stores to the cell surface can thus serve to regulate the pH of synaptic microdomains, which can lend further precision to postsynaptic plasticity during LTP. Indeed, mice deficient in NHE5 displayed improved performance in learning and memory tasks accompanied by an increase in the number of hippocampal excitatory synapses in the hippocampus, which may have arisen from an augmentation in BDNF/TrkB signaling (Chen et al., 2017). Interestingly, past results have also shown that NHE5-mediated pH regulation may also be involved in the surface localization and retrograde signaling through the related tyrosine receptor kinase A (TrkA) (Diering et al., 2013). These data suggest that despite being canonically classified as a plasmalemmal NHE, activity-dependent trafficking of NHE5 to the synaptic cleft may serve to regulate neurotrophin signaling and plasticity. Additional work investigating the importance of NHE5 trafficking and pH regulation will undoubtedly reveal other intriguing roles of this isoform in synaptic physiology and regulation.

INSIGHTS FROM PATIENT-DERIVED NHE6 MUTATIONS
Given the monogenic nature of CS, recent work has also focused upon expressing CS-associated SLC9A6 mutations in both nonneuronal cells and cultured neurons in vitro to investigate consequences to transporter activity and their impact on cell function. Most patient-derived variants harbor non-sense mutations that introduce a premature stop codon in the N-terminal transmembrane region of the protein, thereby preventing synthesis of a full-length protein and complete loss-of-function (LOF) (Gilfillan et al., 2008;Pescosolido et al., 2014). However, several CS-linked variants are missense mutations that result in production of a full-length protein, the effects of which are beginning to be elucidated (Roxrud et al., 2009;Ilie et al., 2014;Ilie et al., 2016;Gao et al., 2019;Ilie et al., 2019;Ilie et al., 2020).
In AP-1 cells [modified Chinese hamster ovary (CHO) cells containing negligible levels of NHE6 protein], most transfected NHE6 variants display impairments in post-translational oligosaccharide maturations and protein misfolding, resulting in enhanced ubiquitination and degradation by the proteasomal (ER-associated protein degradation, ERAD) (Meusser et al., 2005) and/or lysosomal (endosomal sorting complexes required for transport, ESCRT) pathways (Roxrud et al., 2009;Ilie et al., 2014;Ilie et al., 2016;Ilie et al., 2019;Ilie et al., 2020). Moreover, endosomes containing most of these NHE6 mutants are more acidic compared to those expressing NHE6 WT (with some exceptions, see below) (Ilie et al., 2016;Ilie et al., 2020), which is highly suggestive of impairments in endocytic processes. As such, expression of these mutant exchangers in HeLa cells and primary hippocampal neurons also disrupted transferrin uptake (Ilie et al., 2014;Ilie et al., 2016;Ilie et al., 2019;Ilie et al., 2020). These deficits appear to be cargo-selective, as uptake of epidermal growth factor was not disrupted in HeLa cells expressing an in-frame deletion mutant of NHE6 (p.Glu287_Ser288del; ΔES) (Ilie et al., 2016). It should be noted that although both HeLa cells and hippocampal neurons express endogenous NHE6, the introduction of exogenous NHE6 mutants exerts a dominant-negative effect as NHEs typically homodimerize to become functional. Thus, the expression of mutant NHE6 constructs results in the formation of WT and mutant NHE6 heterodimers that perturb the function of the WT fraction of NHE6 (Ilie et al., 2016). This effect is best exemplified in neurons, where transient expression of in-frame deletion mutants ΔES (Ilie et al., 2016) or ΔWST (p.Trp370_Ser_Thr372del) (Ilie et al., 2014) impaired neuronal branching, findings akin to those observed in NHE6 null neurons (Ouyang et al., 2013). Moreover, transfection of exchangerdeficient NHE6 variants into Nhe6 KO neurons failed to rescue neuronal branching (Ouyang et al., 2013), indicating that ion exchange activity of NHE6 is necessary for neuronal morphogenesis. Furthermore, transient expression of some of these mutations into AP-1 cells or primary hippocampal neurons was sufficient to activate apoptotic cell death (Ilie et al., 2016;Ilie et al., 2020). These data strongly suggest that a lack of NHE6 function leads to the pronounced neurodegeneration observed in Nhe6 KO mice (Stromme et al., 2011;Xu et al., 2017) as well as the progressive regression of symptoms in CS patients.
Additional evaluations of the ΔES mutation in cultured hippocampal neurons have recently revealed numerous interesting findings with regards to synaptic function. Neurons expressing ΔES displayed a significant reduction in the number of larger, more mature dendritic spines , again mirroring data from NHE6-deficient pyramidal neurons (Ouyang et al., 2013). Notably, the mutant exchanger was diverted away from EEs and REs towards LEs and lysosomes, which also resulted in a mistrafficking of the AMPAR GluA1 subunit to lysosomes. Consequently, ΔES-transfected neurons were unable to undergo structural and functional remodeling in response to NMDAR-dependent LTP. However, transiently inhibiting lysosomal function or V-ATPase activity was sufficient to partially restore these deficits in synaptic density and plasticity . These results suggest that NHE6mediated pH regulation is necessary to mediate proper AMPAR recycling through the endosomal system, which allows AMPARs to be rapidly inserted into PSDs from intracellular stores during LTP. In the absence of functional NHE6, however, these vesicles become hyperacidified, resulting in the mistrafficking of AMPARs to lysosomes for subsequent proteolysis (Figure 3). This study provides some of the first data showing how excitatory postsynaptic trafficking and remodeling may be impaired when NHE6 function is downregulated. Importantly, it also suggests that reversing dysregulations in vesicular pH-for instance, by using Na + /H + ionophores (e.g., monensin) that mimic NHE6 function (Mollenhauer et al., 1990;Prasad and Rao, 2015) or by employing protonation agents (e.g., chloroquine) that alkalinize endosomal pH (Akpovwa, 2016;Al-Bari, 2017)-may prove to be beneficial therapeutic strategies to treat CS patients (Figure 3). How the ablation of NHE6 function affects synaptic plasticity at the level of the whole circuit remains to be fully elucidated, Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 892196 9 although we predict that functional and structural LTP will be similarly impaired in Nhe6 KO hippocampi.
Parenthetically, certain NHE6 mutants appear to diverge slightly from these patterns. An interesting example of this is the de novo missense NHE6 variant p.Gly218Arg (G218R). When expressed in both AP-1 cells and primary hippocampal neurons, vesicles containing G218R were more alkaline compared to controls . This observation was suggestive of a gain-of-function effect, in contrast to the loss of function associated with the ΔWST and ΔES mutants. While a fraction of G218R was subjected to ERAD, the alkalinization of endosomal pH instead resulted in the trafficking of a significant portion of the mutant exchanger to LEs and extracellular release in exosomes upon LE fusion with the plasma membrane . Nonetheless, neurons expressing G218R showed similar morphological impairments to those expressing LOF mutations, including reduced dendritic arborization and spine density . These results underscore the importance of precisely regulating endosomal pH within strict physiological bounds, as dramatic shifts in either acidification or alkalinization can impose similarly disrupt neuronal and synaptic morphology. This is especially important to consider if endosomal alkalinization agents or genetic rescue approaches are to be used in the treatment of CS, as such therapies will therefore have to be tailored for each specific mutation. In contrast, other mutations identified in CS patients have been demonstrated to exert only negligible effects on exchanger properties and cellular physiology. Specifically, two additional missense mutations, one in the extreme N-terminal cytoplasmic segment (pAla9Ser; A9S) and another in the regulatory C-terminal cytoplasmic domain (Arg568Gln; R568Q) appear to have comparable biochemical characteristics (i.e., biochemical maturation, stability, and vesicular pH regulation) to that of WT NHE6 when expressed in AP-1 cells (Ilie et al., 2020). Engineering the equivalent A9S mutation in mice also revealed normal brain development and neuronal arborization and endosomal pH regulation (Ouyang et al., 2019). In addition, this NHE6 variant appears to be relatively common in healthy individuals (genome aggregation database, gnomAD; RRID:SCR_014964), confirming that this mutation alone does not likely lead to neurological impairments. Conversely, while the R568Q mutation also appeared to be relatively benign in heterologous cells, the effects of this particular variant upon synaptic transmission and plasticity in neurons have yet to be studied. These findings suggest that some patients harbouring NHE6 mutations may have been misdiagnosed, and additional genome-wide analyses will be required to elucidate the true cause of their pathophysiology.
In conclusion, although the prevalence of recorded mutations in both NHE6 and NHE9 continues to increase, treatment options are extremely limited for patients afflicted by neurodevelopmental disorders that arise from altered eNHE function. This paucity arises from an incomplete understanding of the complex roles these eNHEs play at the cellular, molecular and behavioural levels. Even though both NHE6 and NHE9 similarly localize to endosomal compartments, these eNHEs can nevertheless play divergent roles owing to the differences in their spatiotemporal expression patterns. Specifically, the current literature suggests that loss of NHE6 may be especially important during the formation, establishment and potentiation of excitatory synapses in the developing brain, whereas NHE9 may play a greater role in circuit refinement in the postnatal nervous system. This is further reflected in the reciprocity in their expression in ASDs (Schwede et al., 2013), which suggests that NHE9 hyperfunction may also be detrimental to nervous system refinement during adolescence. Moreover, the fact that mutations in NHE6 appear to be much more deleterious than those in NHE9 suggests that the former indeed plays a greater role in excitatory synaptic formation and plasticity, although the contributions of NHE9 within presynaptic terminals and astrocytes should not be discounted. While the growing interest in these exchangers and, more broadly, the role of endosomal dynamics in synaptic function is encouraging, there clearly remains an urgent need to further understand the unique roles of eNHEs in the tripartite synapse. Such knowledge will help guide development of translational strategies to ameliorate the broad range of neurological impairments associated with mutations in these genes.

AUTHOR CONTRIBUTIONS
All authors (AG, EL-D, JO and RAM) contributed to the writing and editing of the manuscript.