Abstract Key Points Question Is NGLY1 Deficiency associated with consistent alterations in brain structure and do these alterations relate to clinical phenotypes? Findings In this case series of 11 patients with NGLY1 Deficiency, MRI brain scans revealed significant subcortical volume reductions across patient age groups, particularly in the thalamus, caudate, and putamen. Younger patients (<3 years old) showed extensive cortical disruptions suggestive of altered gyrification, while older patients (>3 years old) demonstrated region-specific cortical thickness changes. These alterations were associated with several clinical variables, including the presence of seizures, dysphagia, gait disturbance, EEG abnormalities, sleep disturbance, and hearing abnormalities. Meaning Widespread alterations in cortical and subcortical morphology as assessed by MRI highlight the significant impact NGLY1 Deficiency has on brain development. Associations between morphology and clinical variables suggest that these neuroimaging metrics may serve as useful biomarkers of disease progression and in assessing treatment efficacy. Importance NGLY1 (N-Glycanase 1) Deficiency is an ultra-rare autosomal recessive disorder affecting ∼165 patients worldwide, characterized by developmental delay, hyperkinetic movement disorders, and shortened life expectancy. Despite its severe neurological manifestations, comprehensive neuroimaging characterization has been limited to case reports and small descriptive studies. Objective To investigate alterations in brain morphology in patients with NGLY1 Deficiency and determine whether these metrics associate with clinical phenotypes. Design, Setting, and Participants This case series analyzed real-world MRI scans performed on 11 patients with NGLY1 Deficiency between 1999-2023 at sites across the globe. Ages ranged from 2 to 19 years at scan time (5 female, 6 male). Exposure Molecular diagnosis of NGLY1 Deficiency. Main Outcomes and Measures Cortical and subcortical morphology, including subcortical volume, and cortical thickness, surface area, volume, and curvature, were measured with 3-dimensional T1-weighted magnetic resonance imaging (MRI) scans. Z-scores were calculated using normative models from CentileBrain for patients >3 years old or custom models for patients <3 years old. Clinical phenotypes were matched to Human Phenotype Ontology codes. Results 16 scans from 11 patients met quality criteria for analysis. Both age groups (under and over 3 years old) showed significantly reduced subcortical volumes, particularly in bilateral thalamus and putamen. Younger patients demonstrated widespread reductions in cortical surface area, volume, and curvature, indicating altered gyrification patterns. Older patients showed thinner dorsal and thicker ventral cortical regions with limited surface area reductions. Thalamic volume reduction in older patients correlated with gait disturbance, dysphagia, and EEG abnormalities, with additional cortical associations with sleep and hearing abnormalities. Seizure presence in younger patients correlated with altered cortical thickness, surface area, and curvature patterns. Conclusions and Relevance NGLY1 Deficiency is associated with pervasive alterations in brain development affecting both subcortical and cortical morphology. Age-dependent patterns of cortical alterations indicate disrupted neurodevelopmental trajectories that may reflect impaired neuronal migration and/or altered synaptic pruning. Correlations with clinical variables suggest that these measures may serve as useful biomarkers for tracking disease progression and/or treatment efficacy. These findings provide a comprehensive neuroimaging characterization of NGLY1 Deficiency and establish a foundation for understanding brain structure-function relationships in this ultra-rare disorder.

N-glycanase 1 (NGLY1) Deficiency is an ultra-rare autosomal recessive disorder of deglycosylation caused by loss-of-function mutations in the NGLY1 gene. Patients present with developmental delay, intellectual disability, hyperkinetic movement disorder, elevated liver enzymes, (hypo)alacrima, and peripheral neuropathy. Despite supportive care, many experience early neurological deterioration, with loss of previously attained motor skills by adolescence. Additionally, life-threatening complications are not uncommon, and the published median lifespan of patients is 13 years. The pathophysiology of NGLY1 Deficiency remains poorly understood, in part due to limited long-term studies in animal models. Notably, Ngly1⁻/⁻ mice (C57BL/6) are embryonically lethal, and prior characterization of Ngly1⁻/⁻ rats was restricted to young adult rat ( 7 months old), leaving late-onset phenotypes and potential lifespan reduction unexplored. In the study reported here, longitudinal assessments of phenotypes in Ngly1⁻/⁻ rats were conducted alongside Ngly1⁺/⁻ and Ngly1⁺/⁺ control rats. Survival, motor function, biochemical biomarkers, and brain histopathology were examined in the rats from approximately 6 months to 17–18 months of age. Ngly1⁻/⁻ rats exhibited markedly reduced lifespan, progressive neurological decline, and decreased quality of life compared with Ngly1⁺/⁻ and Ngly1⁺/⁺ rats. By 9–10 months of age, 50

GS-100 is an AAV9 gene replacement therapy for the treatment of N-glycanase 1 (NGLY1) deficiency, an ultra-rare, recessive disorder characterized by developmental delay, intellectual disability, hyperkinetic movement disorder, elevated liver enzymes, (hypo)alacrima, and peripheral neuropathy. In pharmacology studies using Ngly1 -/- rats, intracerebroventricular (i.c.v.) administration of GS-100 showed dose-dependent biodistribution, increased hNGLY1 mRNA expression, reduced disease biomarker N-acetylglucosamine-asparagine (GNA), improved motor function, and prevented phenotype progression. Reductions in GNA within central nervous system (CNS) tissues and cerebrospinal fluid (CSF) correlated significantly with improvements, suggesting that CSF GNA may serve as a clinical biomarker of therapeutic activity. Safety studies conducted in wild-type rats and non-human primates (NHPs) indicated no adverse clinical signs, mortality, body weight changes, or negative neurobehavioral/nerve conduction effects at or near the clinical starting dose of 4e14 vector genome (vg)/individual. In addition, GS-100 biodistribution was widespread in CNS and peripheral tissues. These nonclinical data supported the Food and Drug Administration approval of an Investigational New Drug application for a first-in-human phase 1/2/3 dose-escalation clinical trial. The trial's co-primary endpoints include changes in CSF GNA biomarker levels and improvements or stabilization of motor function to assess the safety and efficacy of GS-100 administered i.c.v. in patients with NGLY1 deficiency.

The STING pathway is increasingly recognized as a key regulator of neuroinflammation in neurodegenerative disease, but its role in noninflammatory conditions remains unclear. We generated a postnatal inducible whole-body Ngly1 knockout mouse (iNgly1-/-) to model NGLY1 deficiency, an early-onset neurodegenerative disorder. iNgly1-/- mice exhibit progressive motor deficits, Purkinje cell loss, and shortened lifespan without evidence of gliosis or immune activation. Cell type-specific deletion of Ngly1 in Purkinje cells or microglia failed to induce disease, suggesting multiple cell-intrinsic and cell-extrinsic signals are required. Genetic ablation of Sting1 in iNgly1-/- mice rescues Purkinje cell loss, improves motor function, and extends lifespan. Single-nucleus RNA sequencing reveals proteostasis disruption in Purkinje cells, altered cerebellar granule cell subpopulations, and STING-dependent suppression of cholesterol biosynthesis in glia. Pharmacological inhibition of STING with an orally bioactive antagonist, VS-X4, significantly mitigates neuropathology and motor disease. These findings identify STING as a key mediator of neuropathology in NGLY1 deficiency and implicate a role of STING in noninflammatory neurological disease.

Two new types of novel difuran-coumarin discoid molecule substituted with long-chain alkyl groups were designed and synthesized, which furan-group was successfully coupled with coumarin as the starting material. In this paper, the synthesis method of the peripheral halogenation of novel difuran-coumarin discoid molecules was studied, and its solubility was improved through modification of its periphery, which makes them have high solution processability. And it can expectedly be used as one candidate of organic semiconductor materials.

N-glycanase 1 (NGLY1) Deficiency is a progressive, ultra-rare, autosomal recessive disorder with no approved therapy and five core clinical features: severe global developmental delay, hyperkinetic movement disorder, elevated liver transaminases, alacrima, and peripheral neuropathy. Here, we confirmed and characterized the Ngly1-/-/ rat as a relevant disease model. GS-100, a gene therapy candidate, is a recombinant, single-stranded adeno-associated virus (AAV) 9 vector designed to deliver a functional copy of the human NGLY1 gene. Using the Ngly1-/- rat, we tested different administration routes for GS-100: intracerebroventricular (ICV), intravenous (IV), or the dual route (IV + ICV). ICV and IV + ICV administration resulted in widespread biodistribution of human NGLY1 DNA and corresponding mRNA and protein expression in CNS tissues. GS-100 delivered by ICV or IV + ICV significantly reduced levels of the substrate biomarker N-acetylglucosamine-asparagine (GlcNAc-Asn or GNA) in CSF and brain tissue compared with untreated Ngly1-/- rats. ICV and IV + ICV administration of GS-100 resulted in behavioral improvements in rotarod and rearing tests, whereas IV-only administration did not. IV + ICV did not provide additional benefit compared with ICV administration alone. These data provide evidence that GS-100 could be an effective therapy for NGLY1 Deficiency using the ICV route of administration.

Substrate-derived biomarkers are necessary in slowly progressing monogenetic diseases caused by single-enzyme deficiencies to identify affected patients and serve as surrogate markers for therapy response. N-glycanase 1 (NGLY1) deficiency is an ultra-rare autosomal recessive disorder characterized by developmental delay, peripheral neuropathy, elevated liver transaminases, hyperkinetic movement disorder and (hypo)-alacrima. We demonstrate that N-acetylglucosamine-asparagine (GlcNAc-Asn; GNA), is the analyte most closely associated with NGLY1 deficiency, showing consistent separation in levels between patients and controls. GNA accumulation is directly linked to the absence of functional NGLY1, presenting strong potential for its use as a biomarker. In agreement, a quantitative liquid chromatography with tandem mass spectrometry assay, developed to assess GNA from 3 to 3000 ng/ml, showed that it is conserved as a marker for loss of NGLY1 function in NGLY1-deficient cell lines, rodents (urine, cerebrospinal fluid, plasma and tissues) and patients (plasma and urine). Elevated GNA levels differentiate patients from controls, are stable over time and correlate with changes in NGLY1 activity. GNA as a biomarker has the potential to identify and validate patients with NGLY1 deficiency, act as a direct pharmacodynamic marker and serve as a potential surrogate endpoint in clinical trials.

Located within the brain's ventricles, the choroid plexus produces cerebrospinal fluid and forms an important barrier between the central nervous system and the blood. For unknown reasons, the choroid plexus produces high levels of the protein klotho. Here, we show that these levels naturally decline with aging. Depleting klotho selectively from the choroid plexus via targeted viral vector-induced knockout in Klotho(flox/flox) mice increased the expression of multiple proinflammatory factors and triggered macrophage infiltration of this structure in young mice, simulating changes in unmanipulated old mice. Wild-type mice infected with the same Cre recombinase-expressing virus did not show such alterations. Experimental depletion of klotho from the choroid plexus enhanced microglial activation in the hippocampus after peripheral injection of mice with lipopolysaccharide. In primary cultures, klotho suppressed thioredoxin-interacting protein-dependent activation of the NLRP3 inflammasome in macrophages by enhancing fibroblast growth factor 23 signaling. We conclude that klotho functions as a gatekeeper at the interface between the brain and immune system in the choroid plexus. Klotho depletion in aging or disease may weaken this barrier and promote immune-mediated neuropathogenesis.

Aging is the principal demographic risk factor for Alzheimer disease (AD), the most common neurodegenerative disorder. Klotho is a key modulator of the aging process and, when overexpressed, extends mammalian lifespan, increases synaptic plasticity, and enhances cognition. Whether klotho can counteract deficits related to neurodegenerative diseases, such as AD, is unknown. Here we show that elevating klotho expression decreases premature mortality and network dysfunction in human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Increasing klotho levels prevented depletion of NMDA receptor (NMDAR) subunits in the hippocampus and enhanced spatial learning and memory in hAPP mice. Klotho elevation in hAPP mice increased the abundance of the GluN2B subunit of NMDAR in postsynaptic densities and NMDAR-dependent long-term potentiation, which is critical for learning and memory. Thus, increasing wild-type klotho levels or activities improves synaptic and cognitive functions, and may be of therapeutic benefit in AD and other cognitive disorders.

CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

Aging is the primary risk factor for cognitive decline, an emerging health threat to aging societies worldwide. Whether anti-aging factors such as klotho can counteract cognitive decline is unknown. We show that a lifespan-extending variant of the human KLOTHO gene, KL-VS, is associated with enhanced cognition in heterozygous carriers. Because this allele increased klotho levels in serum, we analyzed transgenic mice with systemic overexpression of klotho. They performed better than controls in multiple tests of learning and memory. Elevating klotho in mice also enhanced long-term potentiation, a form of synaptic plasticity, and enriched synaptic GluN2B, an N-methyl-D-aspartate receptor (NMDAR) subunit with key functions in learning and memory. Blockade of GluN2B abolished klotho-mediated effects. Surprisingly, klotho effects were evident also in young mice and did not correlate with age in humans, suggesting independence from the aging process. Augmenting klotho or its effects may enhance cognition and counteract cognitive deficits at different life stages.

Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including beta-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5(-/-) animals. Loss of SIRT5 leads to accumulation of medium-and long-chain acylcarnitines and decreased beta-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.

In light of the rising prevalence of Alzheimer’s disease (AD), new strategies to prevent, halt, and reverse this condition are needed urgently. Perturbations of brain network activity are observed in AD patients and in conditions that increase the risk of developing AD, suggesting that aberrant network activity might contribute to AD-related cognitive decline. Human amyloid precursor protein (hAPP) transgenic mice simulate key aspects of AD, including pathologically elevated levels of amyloid-β peptides in brain, aberrant neural network activity, remodeling of hippocampal circuits, synaptic deficits, and behavioral abnormalities. Whether these alterations are linked in a causal chain remains unknown. To explore whether hAPP/amyloid-β–induced aberrant network activity contributes to synaptic and cognitive deficits, we treated hAPP mice with different antiepileptic drugs. Among the drugs tested, only levetiracetam (LEV) effectively reduced abnormal spike activity detected by electroencephalography. Chronic treatment with LEV also reversed hippocampal remodeling, behavioral abnormalities, synaptic dysfunction, and deficits in learning and memory in hAPP mice. Our findings support the hypothesis that aberrant network activity contributes causally to synaptic and cognitive deficits in hAPP mice. LEV might also help ameliorate related abnormalities in people who have or are at risk for AD.

The ability to distinguish between similar experiences is a critical feature of episodic memory and is primarily regulated by the dentate gyrus (DG) region of the hippocampus. However, the molecular mechanisms underlying such pattern separation tasks are poorly understood. We report a novel role for the small GTPase ADP ribosylation factor 4 (Arf4) in controlling pattern separation by regulating dendritic spine development. Arf4(+/-) mice at 4-5 months of age display severe impairments in a pattern separation task, as well as significant dendritic spine loss and smaller miniature excitatory postsynaptic currents (mEPSCs) in granule cells of the DG. Arf4 knockdown also decreases spine density in primary neurons, whereas Arf4 overexpression promotes spine development. A constitutively active form of Arf4, Arf4-Q71L, promotes spine density to an even greater extent than wildtype Arf4, whereas the inactive Arf4-T31N mutant does not increase spine density relative to controls. Arf4's effects on spine development are regulated by ASAP1, a GTPase-activating protein that modulates Arf4 GTPase activity. ASAP1 overexpression decreases spine density, and this effect is partially rescued by concomitant overexpression of wildtype Arf4 or Arf4-Q71L. In addition, Arf4 overexpression rescues spine loss in primary neurons from an Alzheimer's disease-related apolipoprotein (apo) E4 mouse model. Our findings suggest that Arf4 is a critical modulator of DG-mediated pattern separation by regulating dendritic spine development.

N-type voltage-gated calcium channels play key roles in many cellular functions including the regulation of neurotransmitter and hormone release. Ca2+ influx through these channels is tightly regulated by second messenger pathways and intrinsic channel properties including voltage-dependent and calcium-dependent inactivation. Less is known about the recovery of calcium channels following inactivation, or if the kinetics of recovery can be modulated by signaling pathways. Recombinant N-type channels (CaV2.2 (α1B), α2δ, and β1b) were expressed in HEK293 cells and barium used as the charge carrier to isolate voltage-dependent inactivation. An individual step depolarization lasting 1-10s, or trains of brief step depolarizations were used to produce inactivation, and recovery was tracked using brief steps at given intervals following the stimulus. Following “short” (<2s) stimuli, recovery was clearly biphasic, but the faster component became minimal after longer duration stimuli (>10s). Acute application of the phorbol ester PMA (50-200nM) had little effect on the resting amplitude of IBa or inactivation during 5-50Hz stimulus trains. However, the slower time constant of IBa recovery was significantly prolonged by PMA. This action of PMA was not blocked by inhibitors of PKC that target the catalytic domain of the enzyme (bisindolylmaleimide I, Go6983, PKC inhibitory peptide 19-36), but was blocked by calphostin-C which targets the diacylglycerol binding C1 domain of PKC and other proteins. Intracellular dialysis with GDP-β-S also blocked the actions of PMA on recovery from inactivation. PMA had a similar effect on endogenous ICa recorded from bovine adrenal chromaffin cells. Our data identify a novel PKC-independent mechanism by which PMA (and perhaps diacylglycerol signaling) modulates the availability of N-type calcium channels during sustained or repetitive activity. Supported by NIH/NINDS (NS052446) and VUMC Discovery grant.

gamma-Secretase cleaves multiple substrates within the transmembrane domain that include the amyloid precursor protein as well as the Notch family of receptors. These substrates are associated with Alzheimer disease and cancer. Despite extensive investigation of this protease, little is known regarding the regulation of gamma-secretase specificity. To discover selective inhibitors for drug development and for probing the mechanisms of gamma-secretase specificity, we screened chemical libraries and consequently developed a di-coumarin family of inhibitors that preferentially inhibit gamma-secretase-mediated production of Abeta42 over other cleavage activities. These coumarin dimer-based compounds interact with gamma-secretase by binding to an allosteric site. By developing a multiple photo-affinity probe approach, we demonstrate that this allosteric binding causes a conformational change within the active site of gamma-secretase at the S2 and S1 sub-sites that leads to selective inhibition of Abeta42. In conclusion, by using these di-coumarin compounds, we reveal a mechanism by which gamma-secretase specificity is regulated and provide insights into the molecular basis by which familial presenilin mutations may affect the active site and specificity of gamma-secretase. Furthermore, this class of selective inhibitors provides the basis for development of Alzheimer disease therapeutic agents.