Creatine plays a vital role in cellular energy production and adenosine triphosphate (ATP) homeostasis and has also been identified as a neurotransmitter in the mammalian brain. Creatine is transported into cells by the human creatine transporter (hCRT) (SLC6A8), an Na+/Cl--dependent symporter encoded on the X chromosome. Mutations in hCRT cause cerebral creatine deficiency syndrome 1, a neurological disorder marked by intellectual disability, speech delay, and seizures. Beyond its role in the brain and muscle, hCRT is highly expressed in metabolically active tumors. Many cancer cells, including colorectal cancer and glioblastoma, upregulate hCRT to sustain intracellular creatine levels and buffer ATP under energy stress. Pharmacological blockade of hCRT by RGX202 has been shown to impair tumor growth by disrupting energy homeostasis. Here, we report the high-resolution cryo-Electron Microscopy (cryo-EM) structures of human hCRT in three states: apo, creatine-bound, and RGX202-bound. hCRT adopts a canonical LeuT-fold with 12 transmembrane helices and two pseudosymmetric inverted repeats. Creatine is coordinated in the central substrate-binding site through interactions with transmembrane helices TM1, TM3, TM6, and TM8, while the inhibitor RGX202 occupies the same binding pocket, engaging in overlapping contacts that competitively block creatine access. Our structural and mechanistic findings clarify substrate recognition and inhibitory binding of hCRT, providing a molecular rationale for targeting hCRT in both inherited metabolic diseases and cancer therapy.

Creatine is a critical metabolite used to buffer cellular energy demands in highly energetic tissues such as the brain and muscle. Genetic defects in endogenous creatine synthesis or transport across cellular membranes lead to a common set of phenotypes referred to as Cerebral Creatine Deficiency Syndrome (CCDS). The most common form of CCDS is Creatine Transporter 1 (CT1) Deficiency (CTD). It accounts for ~ 70% of cases and results from loss-of-function mutations in the X-linked gene SLC6A8. Affected individuals suffer from intellectual disability, autistic-like behaviors, and epilepsy. There are currently no effective therapies for this disorder, but gene therapy has emerged as a potential approach. The two enzymes which comprise the endogenous creatine synthetic pathway (AGAT and GAMT) are selectively expressed by specific cell types throughout the body. However, after synthesized, creatine uptake relies on the protein product of SLC6A8, CT1, to transport creatine into target cell types. We hypothesized that gene delivery of GATM (encoding AGAT) and GAMT into end-user cell types would bypass the need for CT1, allowing for intracellular synthesis of creatine. We tested this strategy in two human cell types: HEK293T cells and primary fibroblasts. Co-delivery of GATM and GAMT increased internal creatine concentrations by 7.6-fold in HEK293T cells and 12.3-fold in healthy control fibroblasts. We then employed this approach to primary fibroblasts from patients with CTD. This resulted in an up to 11.6-fold increase in intracellular creatine concentrations, far exceeding the intracellular concentration of creatine in healthy control fibroblasts. Importantly, overexpression of AGAT and GAMT resulted in proper targeting of these enzymes to their natural cellular compartment and did not impair the growth of patient fibroblasts. These findings establish gene therapy with GATM and GAMT as a potential strategy for patients with CTD.

To summarize the clinical manifestations and genetic characteristics of creatine deficiency syndrome (CDS) caused by GAMT gene mutations. A retrospective analysis was conducted on the clinical and genetic data of two children diagnosed with GAMT deficiency-type CDS at the Children's Medical Center of Xiangya Hospital, Central South University, from December 2020 to December 2024. The two patients presented with symptoms in infancy, and both had compound heterozygous mutations in the GAMT gene. Case 1 exhibited seizures and intellectual disability, while Case 2 had intellectual disability and attention-deficit hyperactivity disorder. Magnetic resonance spectroscopy of cranial MRI in both patients indicated reduced creatine peaks. After creatine treatment, seizures in Case 1 were controlled, but both patients continued to experience intellectual disabilities and behavioral issues. As of December 2024, a total of 21 cases have been reported in China (including this study), and 115 cases have been reported abroad. All patients exhibited developmental delay or intellectual disabilities, with 66.9% (91/136) experiencing seizures, 33.8% (46/136) presenting with motor disorders, and 36.8% (50/136) having behavioral problems. Seventy-five percent (102/136) of patients received creatine treatment, leading to significant improvements in seizures and motor disorders, although cognitive improvement was not substantial. GAMT deficiency-type CDS is rare and presents with nonspecific clinical features. Timely diagnosis facilitates targeted treatment, which can partially improve prognosis. 目的: 总结GAMT基因变异所致肌酸缺乏综合征（creatine deficiency syndrome, CDS）的临床表现及遗传学特点。方法: 回顾性分析2020年12月—2024年12月中南大学湘雅医院儿童医学中心诊治的2例GAMT缺乏型CDS患儿的临床和遗传学资料。结果: 2例患儿均为婴幼儿期起病，GAMT基因均存在复合杂合变异，病例1表现为癫痫发作和智力障碍，病例2存在智力障碍及注意力缺陷多动障碍；2例患儿头颅磁共振波谱提示肌酸峰减低。肌酸治疗后病例1癫痫发作控制，2例患儿仍存在智力障碍及行为问题。截至2024年12月，国内共报道21例患者（包括本研究），国外报道115例。所有患者均有发育迟缓或智力障碍，66.9%（91/136）患者有癫痫发作，33.8%（46/136）患者存在运动障碍，36.8%（50/136）患者有行为问题，75.0%（102/136）患者接受肌酸治疗，癫痫发作及运动障碍可明显改善，认知改善不明显。结论: GAMT缺乏型CDS罕见且临床表现不特异，及时诊断有助于针对性治疗，可部分改善预后。.

Inborn errors of metabolism are a prevalent cause of pediatric neurological abnormalities, often resulting from enzyme deficiencies that disrupt metabolic pathways. Understanding the radiological manifestations of these disorders is critical for timely diagnosis and therapy. This study aimed to elucidate the magnetic resonance imaging (MRI) brain characteristics and magnetic resonance spectroscopy (MRS) findings of specific treatable pediatric neurometabolic disorders through clinical vignettes, thereby enhancing diagnostic accuracy and informing therapeutic approaches. The study includes four cases with varying presentations, all confirmed by genetic testing, and utilized magnetic resonance imaging and spectroscopy to identify characteristic features. The first case, diagnosed as thiamine metabolism dysfunction syndrome type 4, exhibits bilateral corpus striatum T2 hyperintensities with diffusion restriction on MRI brain, increased lactate peak, and reduced N-acetylaspartate (NAA) on magnetic resonance spectroscopy (MRS), and SLC25A19 gene mutation on genetic analysis - features consistent with biotin-thiamine-responsive basal ganglia disease. The second case, identified as thiamine metabolism dysfunction syndrome type 2, reveals T2 hyperintensities in the bilateral corpus striatum, medial thalami, and cerebellar white matter, along with restricted diffusion, reduced NAA, and an inverted lactate doublet on MRS, with genetic analysis confirming an SLC19A3 mutation, also falling under the spectrum of biotin-thiamine-responsive basal ganglia disease. The third case, representing cerebral creatine deficiency syndrome type 2, demonstrates bilateral symmetric T2 hyperintensities in the globus pallidus and central tegmental tracts, a characteristic absence of a creatine peak on MRS, and a confirmed guanidomethyl transferase (GAMT) deficiency gene mutation. The fourth case, diagnosed as hypermanganesemia with dystonia type 2, is characterized by T1 hyperintensity and T2 hypointensity involving the bilateral globus pallidi, substantia nigra, and dentate nuclei, with genetic testing revealing an SLC39A14 mutation. The study emphasizes the importance of MR imaging and spectroscopy in identifying pediatric neurometabolic diseases that can be treated.