DHDDS-related juvenile parkinsonism is caused by impaired lipid metabolism, glycosylation, and mitochondrial dysfunction, which can be rescued by NAD⁺ treatment.

Technical Summary: DHDDS-Related Juvenile Parkinsonism and NAD⁺ Treatment

Problem Statement
Mono-allelic variants in Dehydrodolichyl Diphosphate Synthase (DHDDS) cause a progressive neurological disorder characterized by juvenile parkinsonism, seizures, developmental delay, and mixed movement abnormalities. While DHDDS is a catalytic subunit of the cis-prenyltransferase complex essential for synthesizing dolichol (a lipid carrier for protein glycosylation), the pathophysiology of heterozygous variants remains poorly understood. Unlike recessive forms where glycosylation defects are prominent, heterozygous cases often present with normal blood transferrin profiles, yet exhibit progressive neurodegeneration. Previous hypotheses suggested defective glycosylation, lysosomal dysfunction, and cholesterol accumulation, but these mechanisms had not been fully elucidated in human neuronal tissue, and no disease-modifying therapies exist.

Methodology
The study employed a multi-modal approach combining patient-derived cellular models, high-throughput screening, and clinical observation:

1. Patient Cohort: Six individuals with heterozygous DHDDS variants (including p.Arg205Gln, p.Arg37His, and p.Arg37Cys) were enrolled.
2. In Vitro Modeling: Cortical Brain Organoids (CBOs) and fibroblasts were generated from patient-derived induced pluripotent stem cells (hiPSCs). These models were cultured for up to 150 days to assess developmental and degenerative phenotypes.
3. Multi-Omics Profiling:
   • Lipidomics: Quantified cholesterol, gangliosides (GM1), and dolichol species across developmental timepoints (days 90 and 114).
   • Proteomics & Glycoproteomics: Analyzed protein expression and N-glycosylation patterns at days 60, 90, and 120 to identify pathway disruptions.
   • Mitochondrial Function: Assessed via Seahorse respirometry (oxygen consumption rate) in both fibroblasts and organoids.
   • Electrophysiology: Multi-Electrode Array (MEA) recordings measured network bursts, spike synchronicity, and axon conduction velocity.
4. Drug Screening: A humanized yeast model expressing the pathogenic p.Arg205Gln variant was screened against an ~8,400 compound library to identify growth rescuers.
5. Intervention:
   • In Vitro: CBOs were treated with Nicotinamide Mononucleotide (NMN) at 0.5 µM and 1 µM.
   • Clinical: An off-label, N-of-1 observational trial was conducted where six patients received oral NMN (250 mg/day) for 6 months. Clinical outcomes were measured using the International Co-ordination Rating Scale (ICARS) and video-based analysis of tandem walking and spiral drawing.

Key Results

• Progressive Phenotype in Organoids: Patient-derived CBOs appeared morphologically normal initially but showed signs of degeneration (cell shedding, altered morphology) after 3–4 months. This was accompanied by a significant loss of deep-layer neurons (CTIP2+) by day 150, while early neuronal development (KI67+) was reduced as early as day 60.
• Cell-Type Specific Metabolic Defects:
  • Cholesterol: Significant unesterified cholesterol accumulation was observed in patient fibroblasts and, crucially, specifically in astrocytes (GFAP+) within organoids, but not in neurons.
  • Dolichol Depletion: Lipidomics revealed a progressive, age-dependent depletion of dolichol and related polyprenol species in patient organoids, contrasting with increasing levels in healthy controls.
  • Glycosylation: Glycoproteomics identified progressive abnormalities in N-glycosylation, particularly affecting proteins involved in lipid metabolism (APOH) and lysosomal trafficking (SCARB2/LIMP2).
• Mitochondrial Dysfunction: Both fibroblasts and organoids exhibited significantly reduced maximum and spare respiratory capacity.
• Electrophysiological Abnormalities: Patient organoids displayed increased asynchronicity, with a higher proportion of spikes occurring outside of network bursts.
• NMN Rescue (In Vitro): Treatment with NMN significantly improved mitochondrial respiration (maximal and spare capacity) and normalized the proportion of spikes within network bursts in patient organoids. NMN also reduced cholesteryl ester accumulation but did not restore dolichol levels.
• Clinical Improvement: In the N-of-1 trial, all six treated patients showed clinical improvement after 6 months of NMN supplementation. ICARS scores decreased significantly (indicating improvement), with notable reductions in ataxia, tremor, and rigidity. Video analysis confirmed improved smoothness of movement and reduced deviation during tandem walking.

Significance and Claims
The paper claims to provide the first demonstration that heterozygous DHDDS variants cause progressive glycosylation abnormalities and lipid dysregulation in human neuronal tissue, challenging the assumption that these defects are restricted to recessive forms or non-neuronal tissues. The study identifies a novel mechanism where astrocyte-specific cholesterol accumulation and progressive dolichol depletion contribute to neuronal degeneration.

Crucially, the authors claim that NAD⁺ augmentation via oral NMN supplementation can partially rescue mitochondrial dysfunction and electrophysiological deficits in cellular models and leads to measurable clinical improvements in patients. This suggests that metabolic modulation, specifically targeting NAD⁺ pathways, represents a viable therapeutic strategy for DHDDS-related disease, a condition previously lacking disease-modifying treatments. The findings also highlight the utility of brain organoids for modeling progressive neurodegenerative disorders that are not fully recapitulated by existing animal models.