Surveying Glial Disparities in Two Rare and Neglected Diseases: Non-Ketotic Hyperglycinemia and Kabuki Syndrome

Studying rare neurodegenerative diseases presents significant challenges due to limited epidemiological data, incomplete understanding of pathophysiological mechanisms, and low research investment. In the United States, a disease is classified as rare if it affects fewer than 200,000 individuals – approximately 1 in 2,000 people in a population of 330 million. In this thesis research, I examined two rare disorders: Non-Ketotic Hyperglycinemia (NKH) and Kabuki Syndrome (KS). Both conditions are associated with abnormalities in glial cell populations and result in impaired neurodevelopment. My findings indicate that astrocyte function in NKH and microglial activity in KS are significantly suppressed in their respective mouse models. Both cell types exhibit functional recovery following treatment. These results potentially reveal novel pathophysiological mechanisms in both NKH and KS that have not been previously described. NKH is an autosomal recessive, mitochondrial disorder caused by defects in the glycine cleavage system (GCS) that results in the elevation of glycine both systemically and in the central nervous system. Glycine decarboxylase (GLDC, also called P protein) is a major component of the GCS. Mutations in Gldc cause over 80% of NKH cases. The current estimated prevalence is ~1:76,000 births, with some regional variations; for instance, within the Amish community, the estimate is at ~1:2,000. Clinical presentation is highly heterogeneous with broad, neurometabolic range and is often severe. Adjunctive treatment of glycine reduction frequently fails to confer neurological benefit. In our study, we used an attenuated CRISPR Cas9-edited humanized mouse expressing a prevalent clinical mutation, administered with a single intraperitoneal dose of a novel recombinant of adeno-associated viral vector 9 and GLDC (rAAV9-GLDC). Mice were assessed over five and ten months and validated by assessing the brain and liver. Our control was a single intraperitoneal dose of rAAV9 containing green fluorescent protein (GFP). Our results present a significant increase in astrocytes with minimal inflammatory distress. Additionally, whole brain protein analysis revealed increased levels of neurons and neuronal precursors. However, there were no changes in oligodendrocytes and microglia. Overall, our novel single dose gene therapy increased astrocyte proliferation and eliminated long term neurological defects and death. KS is a rare autosomal heterozygous intellectual disability disorder caused by defects in lysine-specific methyltransferase 2D protein (KMT2D), which results in a decrease in open chromatin mark H3K4me3. Mutations in Kmt2D cause over 55-80% of KS cases. The current estimated prevalence of KS is ~1:86,000 births, with some regional variations; for example, in Japan, the estimate is ~1:32,000. A decrease in open chromatin and trimethylation of H3K4me3 leads to reduced hippocampal functions, low proliferation levels of microglia, and overall cerebral function. To alleviate symptoms, we introduced a triple combination formulation (TCF) that delivers the HDACi vorinostat (Vo) to the brain, bypassing the blood brain barrier (BBB). TCF administration increased the acetylation at H3K4me3, which resulted in promoting microglial arborization, proliferation, and surveillance phenotype. Our results point to an unprecedented defect in KS and provide an effective method of microglial differentiation without a major inflammatory response. My research highlights previously unrecognized glial cell dysfunctions in NKH and KS, offering new insights into the underlying pathophysiology of these rare neurodevelopmental disorders. By demonstrating that targeted therapeutic interventions can restore astrocyte and microglial function without provoking significant inflammatory responses, these findings open new avenues for the development of effective treatments. Overall, this work emphasizes the importance of glial cells in rare neurological diseases and supports further exploration of gene therapy and epigenetic modulation as feasible strategies for addressing unmet clinical needs in these patient groups.

研究罕见神经退行性疾病往往面临多重严峻挑战，包括流行病学数据匮乏、病理生理机制认知不全以及研究投入不足。在美国，若某疾病受累人数少于20万，则被归类为罕见病——对应3.3亿总人口中约每2000人中即有1人患病。本论文研究考察了两种罕见神经发育障碍：非酮症高甘氨酸血症（Non-Ketotic Hyperglycinemia, NKH）与歌舞伎综合征（Kabuki Syndrome, KS）。两种疾病均与胶质细胞群体异常相关，并可导致神经发育受损。

本研究结果显示，在各自的疾病特异性小鼠模型中，NKH的星形胶质细胞功能与KS的小胶质细胞活性均受到显著抑制；而经治疗后，这两类细胞的功能均可得到恢复。上述结果或可揭示此前未被报道过的NKH与KS全新病理生理机制。

NKH属于常染色体隐性遗传性线粒体疾病，由甘氨酸裂解系统（glycine cleavage system, GCS）缺陷引发，可导致全身及中枢神经系统内甘氨酸水平升高。甘氨酸脱羧酶（glycine decarboxylase, GLDC，亦称P蛋白）是GCS的核心组分，Gldc基因的突变可导致超过80%的NKH病例。目前NKH的全球患病率估算约为1:76000活产儿，存在一定地域差异：例如在阿米什社区，患病率可达约1:2000。该病临床表现具有高度异质性，神经代谢谱范围广泛且病情往往较重；目前采用的甘氨酸降低辅助治疗通常无法带来神经功能获益。

本研究中，我们使用了携带常见临床突变的减毒CRISPR Cas9编辑人源化小鼠，并给予单次腹腔注射新型重组腺相关病毒9型（recombinant adeno-associated viral vector 9, rAAV9）与GLDC的复合载体（rAAV9-GLDC）。我们对小鼠进行了为期5个月与10个月的随访评估，并通过检测脑组织与肝脏组织验证实验结果。对照组小鼠仅接受单次腹腔注射携带绿色荧光蛋白（green fluorescent protein, GFP）的rAAV9载体。本研究结果显示，星形胶质细胞数量显著增加且炎症应激水平极低。此外，全脑蛋白质组分析显示神经元与神经元前体细胞水平升高；但少突胶质细胞与小胶质细胞的数量未出现明显变化。综上，我们开发的新型单剂量基因疗法可促进星形胶质细胞增殖，并消除长期神经功能缺陷并降低死亡风险。

歌舞伎综合征（KS）是一种罕见的常染色体显性遗传性智力障碍疾病，由赖氨酸特异性甲基转移酶2D蛋白（lysine-specific methyltransferase 2D, KMT2D）缺陷引发，可导致开放染色质标记H3K4me3水平降低。Kmt2d基因的突变可导致55%~80%的KS病例。目前KS的全球患病率估算约为1:86000活产儿，存在地域差异：例如在日本，患病率约为1:32000。开放染色质水平降低与H3K4me3三甲基化水平下调可导致海马功能受损、小胶质细胞增殖能力下降以及整体脑功能异常。

为缓解相关症状，我们开发了三重联合制剂（triple combination formulation, TCF），可绕过血脑屏障（blood-brain barrier, BBB）将组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor, HDACi）伏立诺他（vorinostat, Vo）递送至脑部。TCF给药可提升H3K4me3位点的乙酰化水平，进而促进小胶质细胞的树突分枝、增殖与监视表型。本研究结果揭示了KS中此前未被发现的小胶质细胞缺陷，并提供了一种可有效诱导小胶质细胞分化且不会引发显著炎症反应的方法。

本研究首次揭示了NKH与KS中此前未被认知的胶质细胞功能异常，为这两种罕见神经发育障碍的潜在病理生理机制提供了全新见解。本研究证实，靶向治疗干预可在不引发显著炎症反应的前提下恢复星形胶质细胞与小胶质细胞功能，为开发有效治疗手段开辟了新方向。综上，本研究强调了胶质细胞在罕见神经系统疾病中的重要性，并支持将基因治疗与表观遗传调控作为解决此类患者群体未被满足的临床需求的可行策略。