Investigating the neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

Domain: physics
Published: 2025-11-26
Editorial outcome: minor_revision

Abstract
Neurodevelopmental proteasomopathies are a group of disorders caused by variants in proteasome subunit genes, that disrupt protein homeostasis and brain development through poorly characterized mechanisms. Here, we report 26 distinct variants in PSMC5, encoding the AAA⁺ ATPase subunit PSMC5/RPT6, in individuals with syndromic neurodevelopmental conditions. Combining genetic, multi-omics and biochemical approaches across cellular models and Drosophila, we unveil the essential role of proteasomes in sustaining key cellular processes. Loss of PSMC5/RPT6 function impairs proteasome activity, leading to protein aggregation, disruption of mitochondrial homeostasis, and dysregulation of lipid metabolism and immune signaling. It also compromises synaptic balance, neuritogenesis, and neural progenitor cell stemness, causing deficits in higher-order functions, including learning and locomotion. Pharmacological targeting of integrated stress response kinases reveals a mechanistic link between proteotoxic stress and spontaneous type I interferon activation. These findings expand our understanding of proteasome-dependent quality control in neurodevelopment and suggest potential therapeutic strategies for neurodevelopmental proteasomopathies.

Introduction
The proper structure and function of eukaryotic cells rely on the maintenance of the intracellular proteome composed of thousands of proteins. The dynamic balance between protein synthesis and degradation is orchestrated by the proteostatic network1,2, an integral component of which is the ubiquitin-proteasome system (UPS), that selectively eliminates short- and long-lived proteins, as well as misfolded proteins modified with ubiquitin3. At the very heart of the UPS is the 26S proteasome, a large multi-subunit protease consisting of a 20S core particle and a 19S regulatory particle, comprising a lid and a base3. The lid subunits recognize and bind polyubiquitinated target proteins1, while the base subunits Rpt1-6/PSMC1-6 utilize ATP hydrolysis to deubiquitinate, unfold and translocate substrates into the proteolytic chamber of the 20S core particle, where they are degraded into oligopeptides. These oligopeptides can be further hydrolyzed into amino acids by peptidases, enabling efficient amino acid recycling within the cellular protein synthesis machinery1,3.

The remarkable dynamics and plasticity of proteasomes impart a significant impact on cellular physiology3, particularly in neurons. Neuronal synaptic plasticity relies on continuous proteome renewal, necessitating robust translation at ribosomes and concurrent breakdown of defective ribosomal products2,4. This maintenance of protein homeostasis is particularly challenging in cortical neurons, where synapses may contain hundreds of proteins4. Effective protein clearance by the proteasome is crucial for various neuronal processes, including synaptic remodeling, cell migration, neurotransmitter release, long-term potentiation, long-term depression, and memory formation5,6. Inhibition of proteasome activity has been experimentally shown to disrupt synapse composition, promote the aggregation of polyubiquitinated proteins, and ultimately lead to neurodegeneration6. Similar observations have been made in brain tissues from individuals with neurodegenerative disorders6 and schizophrenia7. Genetic investigations have further unveiled that loss-of-function variants in proteasome genes contribute to the onset and progression of a unique class of neurodevelopmental disorders (NDDs), specifically referred to as the neurodevelopmental proteasomopathies (NDPs)8 or proteasome associated neurodevelopmental disorders. Unlike proteasome associated autoinflammatory syndromes (PRAAS), which are typically caused by recessive 20S proteasome gene variants and characterized by severe autoinflammatory symptoms, NDPs primarily affect the central nervous system (CNS). The inheritance pattern for these disorders can either be dominant, for the majority of variants in PSMD12 [Stankiewicz-Isidor syndrome, MIM: 617516]9,10, PSMC311 and PSMD1112, or recessive, as observed in cases involving PSMB1 [MIM: 620038]13 and PSMC3 [MIM: 619354]14 variants (Supplementary Fig. 1).

In this context, the proteasome subunit PSMC5/Rpt6 is of particular interest due to its role in regulating proteasome activity and synaptic remodeling. As an ATPase, PSMC5 serves as a neuronal sensor that dynamically adjusts proteasome activity and localization to spines in response to protein turnover requirements through phosphorylation by CAMKIIα15,16. The present study further confirms the pivotal role of PSMC5 in neuronal function. We describe 26 variants in PSMC5, the vast majority heterozygous and de novo, in 44 subjects, including 42 unrelated propositi with delayed neurodevelopment and two affected relatives. Functional analyses performed on patient cells and animal models unveiled that PSMC5 alterations lead to a loss of proteasome function, triggering a complex cellular program involving profound remodeling of innate immunity, inflammation, and lipid metabolism. Intriguingly, the integrated stress response (ISR) mediates most of these changes, and potential molecular targets have been identified, holding promise for the development of biomarkers and therapeutic interventions. This study offers critical insights into the multifaceted impact of PSMC5 variants on NDD, advancing our understanding of the intricate cellular mechanisms underlying these conditions.

Results
Rare pathogenic PSMC5 variants cause a syndromic neurodevelopmental disorder
We identified 44 individuals with similar clinical features who carried a rare single nucleotide variant (SNV) or indel in PSMC5. Thirty-three cases were de novo, six heterozygous of uncertain inheritance, two inherited from an affected mother also enrolled, and one with biallelic variation. In total, we identified 26 distinct variants, most of them missense (19/26) impacting regions highly conserved from human to yeast, and four of them recurring (Fig. 1a and Supplementary Fig. 2). They were predominantly located in the “ATPases Associated with diverse cellular Activities (AAA-ATPase)” domain (16/26); the recurring variants NM_002805.6: c.959C>G p.(Pro320Arg) (6/44 subjects) and c.973C>T p.(Arg325Trp) (9/44) were located near the end of the domain (Fig. 1a).

Our three-dimensional structural analysis showed notably that about half of the variants primarily located in the lower ATPase domain interface with neighboring base ATPase subunits PSMC1/Rpt2 and PSMC4/Rpt3, and the substrate in its engaged state (Fig. 1 and Supplementary Fig. S3). They are likely to affect proteasome function in multiple ways, by disrupting proteasomal complex formation, impairing the transition from substrate-free to substrate-engaged states of the 26S proteasome, affecting ATP binding and hydrolysis, hindering substrate processing, or perturbing protein unfolding and subunit incorporation (Fig. 1; Supplementary Figs. 3 and 4). Overall, these findings, detailed in supplemental notes, concur with the simulated classification of the identified PSMC5 variants according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, suggesting pathogenicity for 25/26 variants (Supplementary Table 1).

Abnormal neurodevelopment was observed in all subjects, with global developmental delay present in 42/44 (95%) cases (Fig. 2a; Supplementary Table 2). Neurological manifestations included absence or delay in speech (36/39; 92%), intellectual disability (26/32; 81%), motor delay or impairment (32/41; 78%), abnormal behavior (30/39; 77%, including autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD)), abnormal muscle tone (27/38; 71%), and seizures (14/43; 33%) (Fig. 2a; Supplementary Table 2). Brain magnetic resonance imaging revealed frequent abnormalities (14/29; 48%) ranging from nonspecific myelination delay to brain atrophy and malformations, such as corpus callosum, pons and cerebellar hypoplasia. The phenotype appeared syndromic (Fig. 2a; Supplementary Table 2), with notable non-neurological findings such as ophthalmological anomalies (27/37; 73%), skeletal malformations (26/42; 62%), feeding difficulties (23/41; 56%), cardiac abnormalities (13/41; 32%), hearing loss (11/39; 28%, conductive in five individuals, sensorineural and mixed in one patient each), genital abnormalities (7/41; 17%) and kidney abnormalities (5/37; 14%).

Subjects with PSMC5 variants present a similar facial gestalt
Most affected individuals exhibited abnormal facial shape (32/40; 80%), often accompanied by craniofacial abnormalities, including microcephaly (15/38; 39%) or abnormality of the mandible (14/39; 36%, including micrognathia, retrognathia and prognathia) (Fig. 2b). However, the manifestations were heterogeneous and did not allow conclusive diagnosis of the disorder through standard clinical assessment. Nonetheless, facial analysis with GestaltMatcher17 suggested a recognizable facial gestalt among PSMC5 subjects. A strong facial resemblance was indeed observed between the 15 PSMC5 subjects tested, 90% of subject combinations having mean pairwise distances below the threshold (c = 0.915) (Supplementary Fig. 5). Analysis by pairwise comparison matrix extended to 7459 images from 449 disorders in GMDB revealed that 10 of 15 PSMC5 subjects (S3/10/12/15/16/23/34-37) matched at least another PSMC5 subject with a rank below 50 (Fig. 2c). Subject pairs/trios (S10, S35 and S37), (S12 and S35), and (S3 and S34) were highly similar, with ranks below ten, indicating strong resemblance.

Variants affecting the AAA+ ATPase domain tend to induce a more severe phenotype
Even if a clear genotype-phenotype correlation is difficult to draw because of an important phenotypic variability across subjects, the multisystemic and syndromic characteristics of the disorder are particularly pronounced when variants are located within or in the immediate vicinity of the ATPase domain. This is especially true for the most recurrent variant in our cohort, c.973C>T p.(Arg325Trp), which is more likely to be associated with severe neurodevelopmental impact and heart defects, facial dysmorphic features, hearing loss, visual impairment and feeding difficulties. On the other hand, variants located outside the ATPase domain, affecting the first part of the protein, are frequently associated with short stature (Fig. 2c; Supplementary Table 2).

Psmc5 knockdown (KD) impairs fly reversal learning and alters proteostasis as well as excitatory/inhibitory balance of rat hippocampal neurons
Pan-neuronal ELAV-GAL4 KD of Rpt6, Drosophila melanogaster’s highly conserved orthologue of PSMC5, had no significant effect on classical olfactory learning, during which flies form association between an odor to foot shock (Tukey, p=0.3059; N=4 performance indexes (PI) per group; Fig. 3a). However, Rpt6 KD caused significant defect in the ability to acquire novel association after initial training—a different behavior paradigm known as reversal learning (Tukey, p<0.0001; N=4; Fig. 3b), which is similar to what we previously reported in proteasome subunit gene PSMC3 KD models11. Interestingly, contrary to wild-type (WT) controls, performance after reversal learning did not change significantly at 1 week (6-8 days) and 3 weeks (22–24) (WT Tukey, N=4 biological replicates) (Fig. 3c).

To determine if Rpt6 KD led to uniform impact on behavior, we tested locomotion activity by leveraging the well-established climbing assay in Drosophila18. We observed a different profile for climbing than that for reversal learning in pan-neuronal Rpt6 KD transgenics (Fig. 3d). While reversal learning deficiencies were similar from day 1 day up to 3 weeks, we observed that decline in climbing performance was initially stable for 1 week (Fig. 3e–h) but dropped markedly at 22 days (Fig. 3i, j). On the other hand, WT flies maintained normal performance during the same period.

We studied the effects of Psmc5 loss on primary rat hippocampal neurons by gene KD, using Psmc5-specific shRNA or scrambled shRNA for controls (scrambled Ctrl) along with co-expressed GFP delivered via lenti-virus infection of cells at day in vitro 1 (DIV1). After 14 days in culture, a significant reduction of PSMC5 levels was measured by fluorescent labeling with PSMC5-specific antibodies (Fig. 3k, l). Notably, under control conditions, PSMC5 labeling appeared predominantly in neurons and much less in glial cells.

Psmc5 KD by shRNA significantly increased ubiquitin-positive protein levels throughout the cells, particularly in the soma. This accumulation of ubiquitin-conjugated proteins (Fig. 3m, n), reflecting impaired proteasomal activity, confirms that PSMC5 is essential for maintaining proper proteasomal function and regulating proteostasis in neurons. To assess the general morphology of neurons developing under PMSC5 KD, we measured their dendritic arborization at DIV14. No significant differences were observed in Psmc5 KD neurons compared to controls (Fig. 3o, p). We also measured synapse density of excitatory synapses (visualized using vGlut as excitatory presynaptic marker) and inhibitory synapses (visualized using vGat as inhibitory presynaptic marker). After 14 days, PSMC5 loss led to a significantly reduced number of vGlut positive puncta along primary dendrites (Fig. 3q, s), whereas the number of vGat positive puncta remained unchanged. (Fig. 3r, t). This indicates an excitation/inhibition (E/I) imbalance due to PSMC5 loss in neurons.

Overexpression of PSMC5 affects neuronal morphology in mouse
A subset of identified missense variants was assessed for their impact on neuronal morphology through ectopic expression in primary mouse hippocampal neurons. Neurite length of mouse hippocampal cells was significantly increased during the neurodevelopmental phase (DIV3) by expression of protein product PSMC5-WT compared to empty vector control (Fig. 4a, b), suggesting a potential role of Psmc5 in promoting neurite outgrowth. Overexpression of variants p.(Arg201Trp), p.(Pro320His) and p.(Arg325Trp) yielded the same effect as PSMC5-WT, suggesting that in this assay these variants do not alter the effect of PSMC5-WT expression. By contrast, overexpression of variants p.(Ala202Val), p.(Thr207Met), p.(Pro320Arg) and p.(Ala324Lys360del) (c.970-2A>G) showed reduced neurite length compared to PSMC5-WT (Fig. 4a, b); this effect, similar to the one induced by the empty vector, suggest that these variants have a loss of function (LoF) effect in the assay. Notably, no effect on arborization was induced by PSMC5-WT and none of the variants had impact on arborization compared to the empty vector control (Fig. 4c).

The PSMC5 variants associated with NDD do not equally impact the PSMC5/Rpt6 subunit steady-state expression and subsequent incorporation into 26S proteasomes
To analyze the ability of PSMC5/Rpt6 variants subunits to integrate 26S proteasome complexes, we ectopically expressed 13/26 identified alterations as N-terminally HA tagged-PSMC5 versions in SHSY-5Y neuroblastoma cells, as previously described11.

While the plasmid-driven production of PSMC5 transcripts was similar between the wild-type and the 13 variant cell lines (Supplementary Fig. 6a), steady-state protein expression levels of PSMC5/Rpt6 were differentially impacted by the variants: they were profoundly reduced by p.(Ala202Val) and p.(Pro320Arg), moderately by p.(Pro183Leu), p.(Arg201Trp), p.(Glu250Val) and p.(Arg325Trp), and mildly by p.(Gln221Arg), p.(Gly216Asp), p.(Arg258Trp), p.(Pro320His), and p.(Met368Thr), whereas they were unaltered by p.(Thr207Met) and p.(Asp394Glufs*2) (Supplementary Fig. 6b). Overall, analyses of the 13 PSMC5 variants examined revealed no significant impact on in vitro PSMC5/Rpt6 abundance when compared to control (Supplementary Fig. 6b, right panel), indicating that haploinsufficiency is unlikely the main driver of variant pathogenicity.

We also observed distinct effects of the 13 PSMC5 variants on proteasome assembly in this in vitro assay. Notably, p.(Ala202Val) prevented PSMC5 incorporation into 19S, 26S, and 30S complexes (Supplementary Fig. 6c), due to the instability of the HA-PSMC5/Rpt6 full-length protein (Supplementary Fig. 6c). Conversely, despite their stable expression in SH-SY5Y cells (Supplementary Fig. 6b), p.(Met368Thr) and p.(Asp394Glufs*2) accumulated in 19S precursors and fully assembled 19S particles without forming 26S and/or 30S proteasome complexes (Supplementary Fig. 6c). Unlike variants p.(Gly216Asp), p.(Gln221Arg), p.(Arg201Trp) and p.(Arg258Trp) which showed unchanged incorporation efficiency compared to their wild-type counterpart, p.(Pro183Leu), p.(Glu250Val), p.(Pro320His) and p.(Pro320Arg) only minimally assembled into mature proteasomes, (Supplementary Fig. 6c). Surprisingly, p.(Thr207Met) had a higher propensity to integrate 19S-capped proteasomes than the wild-type. Densitometric analysis showed that the 13 PSMC5 variants studied had similar incorporation into 19S, 26S, and/or 30S complexes compared to their wild-type counterparts (Supplementary Fig. 6c, lower panel). These findings underscore diverse effects of these variants on subunit expression, protein level stability, and incorporation into mature 26S/30S proteasome complexes.

Most PSMC5 variants do not lead to haploinsufficiency but result in severe proteasome assembly defects
In T cells from affected subjects, none of the PSMC5 variants reduced PSMC5/Rpt6 levels compared to controls, except p.(Pro320Arg) (Fig. 5a). These levels remained unchanged with p.(Arg201Trp), p.(Glu250Val) or p.(Arg325Trp), contrary to in vitro findings in SHSY-5Y cells that had suggested variant-induced protein instability (Supplementary Fig. 6b). This discrepancy may reflect full compensation by the wild-type allele, countering haploinsufficiency as a cause of the disorder. It could also imply a greater turnover of PSMC5 in SH-SY5Y cells attributable to protein-level dosage compensation of the excess of subunits produced following PSMC5 overexpression19. Notably, while steady-state PSMC5/Rpt6 levels were similar to controls, T cells with PSMC5 variants exhibited additional lower-migrating PSMC5/Rpt6 species, potentially due to dysregulated PSMC5 transcript splicing (Fig. 5a, lower panel).

In line with in vitro findings, T cells of subjects S25 [p.(Pro320Arg)], S36 [p.(Arg325Trp)], and S37 [p.(Arg325Trp)] exhibited reduced incorporation of PSMC5/Rpt6 into 26S and 30S proteasome complexes, indicated by reduced PSMC5/Rpt6 staining in 19S-capped proteasomes (Fig. 5b), and confirmed by mass spectrometry-based interactome analysis (Fig. 5c, d). However, in vivo/in vitro discrepancies were observed for other variants: (i) p.(Arg201Trp) and p.(Gln221Arg) reduced PSMC5/Rpt6 and PSMA6/α6 incorporation in T cells from S14 (Fig. 5b). Similar to the unique PRAAS case involving a PSMC5 variant reported so far20, an oligogenic mechanism could underlie the disorder of S14, who harbors a second proteasomal variant, p.(Arg90*) in PSMD11, reported pathogenic elsewhere12. This variant is inherited from a moderately affected father, in whom it reduced 30S proteasomal complexes and α6 staining intensity (Fig. 5b) (ii) p.(Glu250Val) did not impair PSMC5/Rpt6 assembly into 26S/30S proteasomal complexes in S15’s T cells (Fig. 5b) but caused significant disruption in SHSY-5Y cells (Supplementary Fig. 6c). Overall, PSMC5 variants show varied effects on proteasome stability and incorporation.

PSMC5 variants profoundly disrupt protein homeostasis and lipid metabolism, and activate mitophagy in T cells from NDD subjects
To assess how PSMC5 variants affect protein homeostasis, we examined whether T cells from individuals with these variants tend to form protein aggregates. We observed increased aggresome formation in samples from S7/14/15/25/36 (Fig. 6a), along with the accumulation of high molecular weight (HMW) ubiquitin-modified proteins in samples from S1/7/14/15/25/36 (Fig. 6b). These findings demonstrate the loss-of-function nature of PSMC5 variants, which prevent T cells from coping with proteotoxic stress.

To explore the cellular repercussions of proteasome dysfunction, we conducted two separate series of proteomic analyses on subject-derived T cells. Both analyses revealed an enrichment in deregulated mitochondrial proteins (Fig. 7a and Supplementary Fig. 7b). Up-regulated proteins included those involved in membrane structure (MT-ATP8, MTX2, MTCH2) and mitochondrial protein homeostasis and assembly (DNLZ, COX19, GTPBP6), while down-regulated proteins were associated with maintaining mitochondrial integrity under oxidative stress or hypoxia (BOLA1, BCL2L2, AK4, GLRX5). Measurement of mitophagy rates using the pH-sensitive MtPhagy dye showed that T cells with PSMC5 variants (S1/7/14/15/25/37) had a higher percentage of lysosomal-targeted mitochondria than controls (Fig. 7e), confirming that proteasome dysfunction is linked to increased mitochondrial removal and damage.

We also noted an enrichment of down-regulated proteins involved in the early stages of ribosome biogenesis, particularly rRNA processing and 90S pre-ribosomes assembly. This was especially pronounced in the second proteomics series (e.g., MAK16, UTP11, UTP25, PUM3, WDR2, DDX18; Fig. 7b) but was also evident, though less marked, in the first series (e.g., WDR36, TBL3, EBP2; Supplementary Table 3d, e). Transcriptomics of T cells from affected subjects further supported an impairment of ribosome biogenesis (Supplementary Fig. 8a).

Since glycerophospholipid metabolism was also found significantly deregulated (Fig. 7b), we performed T cell lipididomics, which showed massively altered lipid distribution in samples from subjects with PSMC5 variants (Fig. 7c). Levels of membrane phospholipids such as phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), or di- (DG) or tri-acylglycerols (TG), were significantly decreased in patients, whereas cholesterol esters were increased by 50% (Fig. 7d).

Individuals harboring PSMC5 variants display sterile type I IFN responses predominantly triggered by the ISR
Referring to the invariably dysregulated type I interferon (IFN) signaling reported in both PRAAS and NDPs, we performed a targeted transcriptomic analysis on a NanoString panel of autoimmune genes that revealed a strongly elevated type I IFN signature in patients compared to controls (Fig. 8a) also confirmed by T cells transcriptomics (Fig. 8b and Supplementary Fig. 8b).

To further corroborate the association between PSMC5 variants and a type I IFN gene signature, we calculated the IFN scores of T cells from S1/14/15/25/36/37 and unaffected controls based on expression of seven IFN-stimulated genes (ISGs: IFIT1, IFI27, IFI44, IFI44L, ISG15, MX1 and RSAD2). All individuals harboring PSMC5 variants showed elevated expression of ISGs compared to controls (Supplementary Fig. 9), resulting in high IFN scores (Fig. 8c). Intriguingly, the father of S14, carrying the heterozygous PSMD11 variant p.(Arg90*) (Supplementary Table 2), exhibited a positive type I IFN score (Fig. 8c), suggesting that proteasome loss-of-function variants consistently lead to type I IFN gene signatures, even in mildly affected subjects.

The established role of protein kinase R (PKR) in initiating sterile autoinflammatory response to proteasome dysfunction21 prompted us to assess its contribution to the initiation of type I IFN responses in T cells from subjects with PSMC5 variant by using C16, a specific PKR inhibitor. The IFN scores from T cells treated with C16 were significantly lower in T cells treated with C16 than in the same T cells exposed to DMSO (controls), confirming PKR’s role in spontaneous ISG induction in this disorder (Fig. 8d, Supplementary Fig. 10). A comparable reduction was observed upon H-151 treatment (Fig. 8d, Supplementary Fig. 10), underscoring an additional contribution from the cGAS-STING pathway, which senses host-derived DNA in this process that may originate from damaged mitochondria in this setting. Of note, the IFN scores exhibited a dramatic decrease upon treatment with JAK inhibitor baricitinib (Fig. 7c, Supplementary Fig. 8), confirming the autocrine/paracrine nature of this phenomenon. Conversely, blocking the UPR with the IRE1 inhibitor 4µ8C did not yield any significant effects (Fig. S8). Inhibition of another ISR kinase, GCN2, with A92 led to a significant reduction in IFN scores for all subjects, emphasizing the key role of ISR in the generation of type I IFN.

The recurrent PSMC5 heterozygous variant c.973C>T p.(Arg325Trp) perturbs the differentiation of induced pluripotent stem cells (iPSCs) into neural progenitor cells
Given the syndromic and multisystem nature of the condition, we reasoned that proteasome dysfunction caused by PSMC5 variants might disrupt early developmental processes. To address this point, we evaluated the impact of variant c.973C>T p.(Arg325Trp), generally associated with a multi-systemic severe phenotype on the differentiation potential of iPSCs. To this end, the three variant iPSC clones PSMC5(+/p.Arg325Trp) edited by CRISPR/Cas9 and their three isogenic WT controls PSMC5(+/p.Arg325Trp) were used as precursors to generate the three germ layers, and neural progenitor cells (NPCs) (Fig. 9a).

Differentiation of PSMC5(+/+) and PSMC5(+/p.Arg325Trp) iPSCs into the three germ layers resulted in the successful generation of ectodermal, mesodermal, and endodermal cells expressing comparable levels of stage-specific differentiation markers: SOX1 (ectoderm), HAND1 and TBXT (mesoderm), and FOXA2 and SOX17 (endoderm), as assessed by RT-qPCR (Fig. 9b). Notably, a slight but non-significant decrease in the ectodermal marker PAX6 was observed in PSMC5(+/p.Arg325Trp) cells compared to PSMC5(+/+) suggesting minimal impact of variant PSMC5 c.973C>T p.(Arg325Trp) on the formation of the three layers. However, a significant down-regulation of HES1 was observed by transcriptomics in PSMC5(+/p.Arg325Trp) ectodermal cells compared to PSMC5(+/+) cells (Fig. 9c and Supplementary Fig. 11). Transcriptomics also showed an increased expression of MSMO1 (Fig. 9c, d), which indicates a likely deregulated cholesterol metabolism (Fig. 9d and Supplementary Fig. 11).

Considering the neurodevelopmental abnormalities observed in all subjects (Fig. 2a), we also assessed the impact of PSMC5 variants on neuronal processes, particularly neurogenesis, by differentiating PSMC5(+/+) and PSMC5(p.Arg325Trp) iPSCs into neural progenitor cells (NPCs). Nearly all day-28 NPCs derived from PSMC5(+/+) iPSCs expressed the neuronal markers Nestin and SOX2 (Fig. 9e, f). In contrast, a significant portion of NPCs derived from PSMC5(+/p.Arg325Trp) iPSCs failed to express these markers, as determined by fluorescence microscopy. Indeed, a thorough analysis of the microscopy images revealed that the proportion of SOX2-positive cells was 10% lower, and the proportion of Nestin-positive cells was 7% lower in PSMC5(+/p.Arg325Trp) NPCs compared to wild-type NPCs (Fig. 9e, g). These results confirm that the PSMC5 c.973C>T p.Arg325Trp variant is associated with impaired neurogenesis. Altogether, these data highlight the detrimental role of proteasome variants in early development, specifically by impairing the genesis of NPCs. These findings establish a clear cause-to-effect relationship between PSMC5 variants and the observed clinical phenotype.

Methods
Details regarding the reagents, equipment, software, oligonucleotide sequences, animal strains, and cell lines used in the following methodological sections are provided in Supplementary Table 5a–e.

Genetic studies and ethics statement
Forty-four affected individuals, along with their healthy parents where possible, were included in this study. Enrollment was conducted by diagnostic or research teams as listed in Supplementary Table 6a. Written informed consent was obtained from all participants or their legally authorized representatives. Each affected individual was clinically evaluated, and written consent for use with the GestaltMatcher tool was secured for subjects S1, S3, S10, S12, S13, S15, S16, S23, S34-38, S40, and S44. Genome or exome sequencing was performed on all affected individuals, typically using a trio-based approach. All data included in this study were collected and shared in compliance with institutional policies and applicable regulations. Centers enrolling patients in a research context obtained approval from their local ethics committees (Supplementary Table 6b). For selected centers contributing data from a single patient in a diagnostic setting, IRB approval was not required, as the data were fully anonymized prior to transfer and excluded any photographic or personally identifiable information.

Three-dimensional (3D) structural analysis of PSMC5 variants
To assess the spatial distribution of amino acid substitutions within PSMC5/Rpt6, we examined two high-resolution cryo-electron microscopy (cryo-EM) structures of the human 26S proteasome: one in a substrate-engaged state (PDB ID: 6MSK) and the other in a substrate-free conformation (PDB ID: 7W37). Structural mapping and visualization of the variants were performed using UCSF ChimeraX.

Facial image analysis
Facial similarity among 15 PSMC5 subjects (S1, S3, S10, S12, S13, S15, S16, S23, S34–38, S43, S44) was assessed using the GestaltMatcher (GMDB) approach17, with parental consent. Each image was encoded via model ensemble and test-time augmentation into twelve 512-dimensional vectors. Pairwise similarity was quantified by averaging 12 cosine distances; lower values indicate greater resemblance.

To evaluate intra-cohort similarity, we compared mean pairwise cosine distances among PSMC5 subjects to two control distributions derived from 1,555 GMDB images (328 syndromes)63: (1) subjects sharing a syndrome, and (2) randomly selected individuals. For each syndrome S and cohort size n (2≤n≤|S|), we sampled 100 cohorts: C (n patients from S) and C (n random GMDB patients), excluding duplicates. Mean pairwise distances were computed for each.

Discriminative power was assessed via Receiver Operating Characteristic (ROC) analysis using mean pairwise distance as the metric, with 5-fold cross-validation. The optimal threshold (c=0.915) was selected via Youden index, yielding sensitivity=0.851, specificity=0.862, and AUC=0.895.

To test PSMC5 cohort similarity, we sampled sub-cohorts C of size n (2≤n≤|C|), computed their mean pairwise distances, and repeated this 10,000 times (duplicates removed) to generate a referenced distribution.

Finally, pairwise comparisons were performed across the 15 PSMC5 subjects. Each was tested via leave-one-out cross-validation against 7459 GMDB images (449 disorders), ranking the remaining 14 PSMC5 subjects within the broader image space.

Behavioral studies in fly
Background. Rpt6, the Drosophila ortholog of PSMC5, shows strong homology (DRSC integrative ortholog prediction tool score 15/16). KD was performed using the GAL4-UAS system, based on truncating patient variants (V5: p.(Lys196Argfs29), V23: p.(Asp394Glufs2); Fig. 1a) predicted to impair proteasome function.

Drosophila strains and crosses. UAS-Rpt6 RNAi, ELAV-GAL4, and wild-type strains were used. Flies were reared on cornmeal agar at 22°C and 40% humidity under University of Alberta biosafety regulations. Virgin ELAV-GAL4 or wild-type females were crossed to UAS-RNAi males.

Olfactory learning. Groups of 100 flies were trained to associate one odor with footshock64, while a second odor was presented without. Learning was assessed by odor avoidance. Reciprocal experiments on 100 naïve flies controlled for odor bias. Performance index was calculated four times from both trials (n=4 performance indexes per biological replicates of 200 flies).

Reversal learning. Flies received an additional training cycle with inverted odor–shock pairings prior to testing, requiring them to unlearn the initial association and acquire a new one. Reversed pairings were used on 100 naive flies to control for bias. Performance index was calculated from both trials (n=4 biological replicates).

Assays were conducted at 25°C, 70% humidity. Adults were transferred to bottles the day before testing and assayed at 1–3 days post-eclosion. Aged flies were maintained on standard food and transferred weekly to avoid contamination with next generation.

Climbing18. Groups of 20 one-day-old flies were placed in vials the day before testing. During trials, flies were tapped to the bottom of a 250ml glass cylinder, and the number reaching 17.5 cm within 2 min was recorded. Results were expressed as the percentage above the line at 10 s intervals (n=10 biological replicates).

Statistics. Two-tailed t-tests were used for olfactory and climbing assays. Reversal learning was analyzed via one-way ANOVA followed by Tukey’s post hoc test. All analyses were performed using Prism.

Psmc5 KD in rat hippocampal neurons
Primary neuron culture. Rat hippocampal neurons were seeded on poly-L-lysine (0.1mg/mL)-coated coverslips in 24-well plates (5×10⁴ cells/well) and cultured for 14 days in Neurobasal medium supplemented with 2% B27, 1% Glutamax, and 100µg/mL penicillin/streptomycin at 37°C, 5% CO₂.

Lentiviral KD. Twenty-four hours post-seeding, Psmc5 shRNA lentiviral particles were added at a multiplicity of infection of 5 for 18 h before medium replacement.

Immunocytochemistry. Neurons were fixed (4% PFA, 15min), permeabilized (0.2% Triton X-100, 5min), and blocked (10% FBS in PBS, 1h). Primary antibodies—MAP2 (1:1000), vGlut (1:1000), vGAT (1:200), ubiquitin (1:500), and PSMC5 (1:400) in blocking solution—were applied overnight at 4°C. After PBS washes (1X, 3×5min), secondary antibodies (Alexa Fluor® 568) were incubated for 1h in the dark. Cells were washed (1X PBS, 2×5min), then nuclei were counterstained with DAPI (300nM, 5min), followed by final PBS and ddH₂O washes. Images were acquired at 40x magnification using an ImageXpress Micro Confocal microscope and analyzed with ImageJ v1.53. Each “n” represents the number of images analyzed per condition in each experiment.

Neuronal morphology analysis in mouse
Cloning and constructs. Human PSMC5 WT (NM_002805.6) and mutant cDNA sequences were PCR-amplified from pcDNA3.1/Zeo(+) expression vector, tagged with Asc/PacI sites, and ligated into a dual-promoter expression vector (CAGG-driven PSMC5 expression; PGK-driven dtTomato for transfection tracking). An empty vector (EV) lacking PSMC5 insert served as a control. The following human PSMC5 variants were introduced in mutant constructs: c.601C>T p.(Arg201Trp), c.605C>T p.(Ala202Val), c.620C>T p.(Thr207Met), c.959C>A p.(Pro320His), c.959C>G p.(Pro320Arg), c.973C>T p.(Arg325Trp), and a deletion mimicking a splice site variant (Ala324Lys360del).

Animals and ethics. FvB/NHanHsd mice were group-housed and crossed under standard conditions with ad libitum food and water. All experiments complied with EU Directive 2010/63/EU (license AVD101002017893) and were approved by the Erasmus MC IRB.

Primary hippocampal cultures. Hippocampi from E16.5 embryos were dissected in ice-cold Neurobasal medium (NB), digested with Trypsin/EDTA (20min, 37°C), and washed in warm NB. Cells were dissociated in supplemented NB (1% penicillin/streptomycin, 1% GlutaMax, 2% B27), seeded on poly-D-lysine-coated coverslips in 12-well plates (1mL/well), and incubated at 37°C, 5% CO₂.

Transfection and immunostaining. Neurons were transfected on DIV3 with 2.5µg DNA per PSMC5 WT or mutant construct or 1.8µg EV using Lipofectamine 2000. Cells were fixed at DIV8 (4% PFA/sucrose), labeled with MAP2 antibody (1:500) and visualized with Alexa 647-conjugated secondary antibody (1:200), and mounted in Mowiol for confocal imaging.

Imaging and analysis. Images were acquired using a Zeiss LSM700 confocal microscope (20× objective, 0.5 zoom, 2048×2048) from 10 neurons per condition per batch. Neurite length and branching were quantified using ImageJ with NeuronJ plugin. Data were normalized to PSMC5 WT neurons and pooled across batches. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test (GraphPad Prism). Each “n” represents a single traced neuron; ≥8 neurons per condition from ≥2 independent batches.

T cell expansion from PBMC
Peripheral blood was collected from healthy donors and Subjects S1/7/14/15/25/36/37, along with available relatives. PBMCs were isolated via density gradient centrifugation and cryopreserved in 90% FBS/10 % DMSO. For expansion, PBMCs were co-cultured with irradiated allogeneic PBMCs in 96-well plates, supplemented with IL-2 and PHA-L (as in ref. 11). Cells were maintained in RPMI1640 with 10% human AB serum and 1% penicillin/streptomycin, and analyzed at rest after 3–4 weeks.

SDS-PAGE and immunoblotting
Resting T cells and/or SH-SY5Y cells were lysed in RIPA buffer; protein concentration was determined via BCA assay. Lysates (20µg) were resolved on 10–15% SDS-PAGE, and transferred to PVDF membranes (wet transfer, 200V, 400mA, 1h, 4°C). Membranes were blocked (ROTI® Block, 20min), incubated overnight at 4°C with primary antibodies targeting HA, PSMC5/Rpt6, α-tubulin, α6, K48-linked ubiquitin, GAPDH, GRP94, ATF6, BNIP3L. Detection was performed using HRP-conjugated secondary antibodies and chemiluminescence.

RNA isolation, reverse-transcription and PCR analysis
Total RNA was extracted from snap-frozen SH-SY5Y and T cells, and 500ng was reverse-transcribed (M-MLV). Real‑time PCR was performed in duplicates to quantify ISGs (IFIT1, IFI27, IFI44, IFI44L, ISG15, MX1, RSAD2) and GAPDH using FAM‑tagged TaqMan™ assays. Relative expression was calculated via the 2−ΔΔCt method. IFN scores were derived as the median RQ of the seven ISGs relative to a calibrator (as in ref. 11). Semi-quantitative PCR on SH-SY5Y cDNA amplified HA-PSMC5 using primers targeting PSMC5 and the polyadenylation signal of the pcDNA3.1/Zeo(+) vector. Products were resolved on 1.7% agarose gels, stained with GelRed®, and visualized under UV (312nm).

Native-PAGE and in-gel activity assays
Resting T cells from control and NDD patients with PSMC5 variants underwent three freeze‑thaw cycles (liquid N₂) in TSDG buffer. Supernatants were collected post-centrifugation (14,000×g, 15min, 4°C) and protein quantified via Bradford assay. Extracts (20µg) were resolved on 3–12% Bis-Tris gels; proteasome activity was assessed by incubating gels with 0.1mM LLVY-AMC at 37°C for 30min. Gels were transferred to PVDF membranes and probed with antibodies against PSMC5/Rpt6 and α6. Detection was performed using HRP-labeled secondary antibodies and chemiluminescence.

Proteostasis and mitophagy assays
T cells expanded from PBMCs of NDD subjects S7, S14, S15, S25 and S36 were stained with 1µM PROTEOSTAT® dye and analyzed using the B3 (PerCP-Vio700) channel (Supplementary Fig. 13).

In parallel, T cells expanded from subjects (n=7; S1/7/14/15/25/36/37) and healthy donors (n=7) were incubated overnight with 100nM Mtphagy dye and analyzed by flow cytometry using the B4 (PE-Vio770) channel.

Lipid profile analysis
Lipid profiling was performed by high-resolution mass spectrometry coupled to reversed-phase UHPLC. Lipids were extracted from donor-derived PBMCs following a protocol slightly adapted from ref. 65. Briefly, 225μL cold MeOH (0.01 % BHT) was added to cell pellets, vortexed, pipettied, and sonicated (30s, on ice). After addition of 3μL EquiSPLASH™ and 750μL cold MTBE, samples were incubated 1h at 4°C (650rpm). Following addition of 188µL ultrapure water and centrifugation (10,000×g, 10min), the upper organic phase (700µL) was collected. The lower phase was re-extracted with 400µL MTBE and 10µL acetic acid (30min, 4°C), and 400µL of the organic layer was pooled. After drying over sodium sulfate, solvents were evaporated under nitrogen and samples stored at –80°C.

Dried extracts were rehydrated in chloroform/methanol/isopropanol (1:2:4, buffered with ammonium acetate pH 8) and analyzed in duplicate using a Vanquish UHPLC coupled via nano-electrospray to a QExactive Plus mass spectrometer (positive/negative mode, four injections/sample). Separation was achieved at 50°C using a linear gradient from buffer A (60:40 water:acetonitrile, 10mM ammonium formate, 0.1% formic acid) to buffer B (90:10 isopropanol:acetonitrile, same additives), ramping from 20% to 99% B over 29min. Spectra were acquired in DDA mode (loop count 15) with 70,000 resolution (MS1) and 17,500 (MS2); collision energy was set to 25. Spray voltage (HES III) was 2.80 kV with polarity-specific adjustments.

Raw data were processed using LipidSearch software with precursor/product tolerances of 5/7 ppm, m-score ≥ 2.0, and filters for top rank, main isomer peak, fatty acid priority, and ID quality A–B (Identification is limited to the full structure, e.g., head group, glycerol backbone, fatty acids). All lipid classes were included. Ion adducts: +H, +NH₄, +2H (positive); –H, +HCOO⁻, –2H (negative). Identified species were filtered by peak quality (>0.8) and mass deviation (–1 to +1 ppm).

Data were subsequently processed parsed in R (tidyverse); intensities were normalized to EquiSPLASH internal standards, technical replicates averaged, and samples median–median normalized with half-minimum imputation. Principal component analysis (PCA) was performed using the R-packages FactoMineR and factoextra. Lipid class fold changes were calculated as ratios of mean lipid ion intensities of the patient group compared to the healthy donor group. Statistics were calculated using Student’s t-test.

NanoString profiling
Gene expression was assessed from 100ng RNA extracted from control and affected T cells using the NanoString nCounter® Human AutoImmune Profiling Panel, following manufacturer’s instructions. Data were normalized to housekeeping genes accordingly.

iPSC generation and differentiation
Generation of variant iPSC lines. To assess the impact of the recurrent PSMC5 variant c.973C>T p.(Arg325Trp), we introduced it by genome editing in the commercial iPSC line ASE-9211, devoid of any variants in UPS genes. CRISPR/Cas9 editing was conducted via nucleofection (4D-Nucleofector™, Lonza) of 1×10⁶ iPSCs with 200pmol HDR template and a ribonucleoprotein complex (225 pmol crRNA, 225 pmol tracrRNA-ATTO+, 120 pmol Cas9; IDT). After 24h, ATTO+ cells were sorted by FACS (MoFlo Astrios, CYTO-ICAN, Paris) and plated at low density (40–50 cells/cm²) on Synthemax II substrate with CloneR™ 2 supplement for clonal selection. Clones were picked after 7 days, expanded on Laminin-521, cryopreserved, and genotyped by PCR/Sanger sequencing to confirm variant incorporation. Chromosomal integrity was assessed using the iCS-digital™ PSC kit.

iPSC culture and trilineage differentiation. All procedures involving genetically modified organisms were approved by the French Ministry of Higher Education and Research (Article L.532-3). iPSCs were cultured in mTeSR™ 1 medium on Matrigel® hESC-qualified matrix. Trilineage differentiation was performed using ASE-9211, two edited WT clones (C10, E5), and three variant clones (G1, G8, H1) with the StemMACS™ Trilineage Kit. After 7 days, RNA was extracted, and 1µg was reverse-transcribed. qPCR was performed using TaqMan assays for SOX1, PAX6, HAND1, TBXT, FOXA2, and SOX17.

NPC differentiation. NPCs were generated over 28 days using the STEMdiff™ Neural System from ASE-9211, three WT clones (B10, C10, E5), and three variant clones (G1, G8, H1). On day 28, cells were seeded on coverslips (5×10⁵ cells/well), fixed (4% PFA), permeabilized (0.5% Triton X-100), and blocked (0.5% BSA). Immunostaining was performed with anti-SOX2 and anti-Nestin antibodies, followed by Alexa Fluor® 488/568-conjugated secondary antibodies (1:1,000). Nuclei were counterstained with DAPI (300nM), and cells were imaged at 10× magnification (Nikon Eclipse Ti2). SOX2/Nestin-positive cell frequencies were quantified using ImageJ.

Transcriptomics
Data generation. Two cell types were analyzed: (i) T cells from 12 healthy donors and four affected individuals (S15, S25, S36, S37); after culture, 1×10⁶ cells were lysed in 700µL Qiazol and RNA extracted using the miRNeasy kit. (ii) Ectodermal cells derived from six iPSC lines: three with p.(Arg325Trp), two isogenic WT controls, and the commercial line ASE-9211, differentiated using the trilineage kit.

RNA quantity and integrity were assessed via Qubit-Flex fluorometer and LabChip GX Touch bioanalyzer. Libraries were prepared from 10ng RNA using 3′ digital gene expression profiling (as in ref. 66), incorporating universal adapters, barcodes, and unique molecular identifiers (UMIs) during template-switching reverse transcription. Barcoded cDNAs were pooled, amplified, and fragmented to enrich 3′ ends. Libraries (350–800bp) were sequenced on a NovaSeq 6000.

Bioinformatics. Primary analysis is detailed in Supplementary Information. Differential expression was performed using DESeq268 with pre-filtering (counts <10 removed), reducing genes from 28,307 to 15,990. Wald test identified differentially expressed genes (adjusted p≤0.05, Benjamini-Hochberg correction, log₂FC≥0). Pathway enrichment was conducted using clusterProfiler; figures were generated with ggplot2 in R.

Proteomics of T cells (second set of analyses)
20S proteasome purification. T cells from 13 individuals (n=13; four patient samples, including three separate samples for S14, S15, S25, plus a pooled sample for S36 and S37; nine healthy donors) were expanded from PBMCs (80–100×10⁶ cells/sample), lysed in HEPES buffer (10mM HEPES pH 7.9, 1% NP40, 10mM KCl, 10mM EDTA, 10mM ATP, 5mM MgCl₂, protease/phosphatase inhibitors), sonicated (15 cycles, 30s on/off, Bioruptor Pico), and centrifuged (14,000×g, 30min, 4°C). Supernatants (1–5mg protein) were incubated overnight with MCP21 antibody (50µg, anti-α2 subunit) cross-linked to Protein G MagBeads (50µl 25% slurry) using 20mM dimethyl pimelimidate in 0.2M triethanolamine (pH 8.2). Beads were washed (Tris buffer with ATP/MgCl₂) and eluted in 5% SDS/50mM ammonium bicarbonate (50µL). Eluates were snap-frozen and stored at –70°C for LC-MS/MS. Aliquots (20µL) were retained for global proteome analysis.

Bottom-up LC-MS/MS and data processing. Lysates/eluates from protein samples of 13 individuals (n=11; 5 patients; 6 healthy controls) were reduced (100mM TCEP), alkylated (400mM CA, 95°C, 5min), digested with trypsin (2% protein weight, ≥1µg) using S-Trap™ micro spin columns, and desalted using Evotip. Peptides (2µg) were analyzed by nanoLC (UltiMate 3000 RS, Thermo) coupled to TimsTOF SCP MS (Bruker) on a C18 Aurora column (25cm×75µm, IonOpticks). Gradient: 2–20% B (30min), 20–37% (3min), 37–85% (2min); flow rate: 150nL/min. DIA-PASEF acquisition covered 400–1,000m/z and 1/K₀ 0.64–1.37, with 8 TIMS ramps (100ms accumulation, 3 MS/MS windows of 25Th). Collision energy ranged from 59eV (1/K₀=1.6) to 20eV (1/K₀=0.6).

Raw data were processed with DIA-NN 1.9 using a predicted UniProt human library including PSMC5 variants and contaminants67. Results were validated in Proline68 (FDR≤1%, peptide length 7–30, charge 2–3). Significant peptides/proteins were identified using t-tests (p<0.01), median ratio fitting, and normalization. Missing values (≤2) were imputed using the 5% centile; t-tests and z-tests were applied.

Global proteome analysis. Contaminant libraries were used to exclude background signals67. Protein abundances were normalized globally. Differential expression was visualized via volcano plots (p<0.05, log₂FC≥±1) using ggplot2. Mitochondrial proteins were annotated using MitoCarta 3.0 inventory69.

Proteasome interactome analysis. IBAQ values were calculated by dividing protein abundance by observable peptides, normalized to total 20S content (average IBAQ of PSMA1–7, PSMB1–4). Stoichiometry and relative abundance of 20S subunits, activators, and interactors were computed as per Fabre et al.70. Comparisons used RM one-way ANOVA with Geisser–Greenhouse correction, followed by Šidák’s multiple comparisons test with individual variance estimates.

Statistics and reproducibility
No statistical method was used to predetermine sample size. No data were excluded from the analyses, except for proteomics (second set of analyses) outliers with a high proportion of missing values. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review of paper by Kury et al. entitled Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.\nIn this manuscript the authors identified 23 unique variants in PSMC5 in 38 unrelated NDD individuals. The authors used a lot of different techniques and animal models (fly-mouse-rat) but to my opinion, these experiments were to superficial and not helping to elucidate the underlying general pathophysiology in the CNS.\nThe data obtained in the human T cells, on the other hand, is much more relevant and robust, but still do not fully explain the variety of clinical symptoms. I am missing the actual fundamental link between these mutations and the pathology.\nMajor remarks\nSeveral mutations were “assumed de novo”. What does this mean exactly? Were parents tested yes or no? This is a very vague term to me.\nAlmost all patients carry a missense mutation. Patient S9 is homozygous for the mutation p.Thr207Met. This missense is also located in the AAA+ ATPase core domain, so one would expect that the heterozygous parents would also exhibit symptoms? Were they clinically examined, also for more subtle symptoms. It would also be interesting to take their T-cells along for the functional analyses.\nIs there actually an average facial gestalt?\nGiven the dysregulation in type I IFN production, one would expect (auto)-immune problems. How thoroughly were the patients investigated for this? Any problems in the carrier parents of patients S5 and S17? They should be investigated.\nThe same holds true for the role of the proteasome in age-related neurodegenerative diseases. What is the age of the carrier mothers of patients S5 and S17? Were they investigated? Was segregation analysis done in these families? Maybe there are other family members that are older and carrier of the mutation.\nDo proteasome-linked genes, and PSMC5 in particular, pop-up in GWAS studies?\nMinor remarks\nL 268: delete second “in this context”\nL 653: day one of development ? To what exact developmental time point does this correspond?\nIn the abstract, the authors state that they looked in “human” hippocampal neurons? Typo probably?", "reviewer_2": "Authors describe a total of 23 pathogenic PSMC5 variants in 38 individuals of neurodevelopmental disorders with various other organs involvement. PSMC5 encodes the AAA-ATPase proteasome subunit PSMC5/Rpt6, a component of the 26S proteasome complex. Several multimodal functional analyses have been performed: PSMC5 overexpression resulting in altered human hippocampal neuronal morphology, PSMC5 knockdown causing impaired reversal learning in Drosophila and loss of excitatory synapses in rat hippocampal neurons, abnormal proteasome assembly defects associated with most PSMC5 variants, abnormalities of innate immune signaling, mitophagy and lipid metabolism together with their witness of PKR and GCN2 kinases potentially ameliorating immune dysregulations in affected patients. This reviewer thinks identification of 23 novel variants and various functional assays are well-done, providing new scientific aspects in the new proteasomopathy, and suggest several points which may improve the manuscript. Recently, neurodevelopmental proteasomopathies with abnormalities of PSMD12, PSMC3, and PSMB1 have been reported, and PSMC5 abnormalities were added by this manuscript.\nMajors\n• Since the impact of each variant is complex and varies depending on the target of observation (e.g., effects on PSMC5 stability, proteasome assembly, mitophagy, IFN signature, etc), it is easier to understand if functional impacts by pathogenic variants are summarized in a figure or table.\n• A scheme summarizing the causative genes and phenotypes of proteasome-related neurodevelopmental proteasomopathies and diseases with similar cellular dynamics to this disease, such as CANDLE and PRAAS, would be helpful for the understanding of readers.\n• Are there any phenotype-genotype correlation among the position of variants regarding 3D structure, functional domains, in vitro phenotypes?\n• Two affected mothers of S5 and S17 should be characterized at the similar level to their children. Perhaps include such detailed information in Tables 1, 2 and S2.\n• Parents of S9 with a homozygous variant are assumed to have the variant heterozygously. Even if DNA is unavailable, their phenotypic information should be helpful to consider how the homozygous variant should be positioned in this manuscript. Please comment on the phenotypic difference between heterozygosity and homozygosity. Is this family consanguineous?\n• Facial image analysis using GestaltMatcher is interesting. Please comment of the most typical face of PSMC5 variants if possible. S9 is a homozygous patient and others are all heterozygous patients, though.\n• S26 with p.(Pro320His) has a twin with similar presentations and ADHD in the father. How about their genetic inheritance? The allele count for gnomaAD also has 8 alleles.\n• Different results between SHSY-5Y cell experiments and patients’ T cells was considered to be due to compensatory effects. But this could be due to difference cell natures.\n• Increased LMNA expression may cause premature aging. How about the accumulation of falnesylated pre-laminA (progerin)?\n• Why do patients with PSMC5 variants show no obvious signs of autoinflammation?\n• Are there any neurological symptoms that appear in late onset due to accumulation of ubiquitinated proteins in older patients?\n• Since PSMC5 function may differ depending on the time of development and tissues, it is preferable to evaluate the pathophysiology using not only cultured cells but also knock-in mice.\n• Are inclusion bodies composed of ubiquitinated abnormal proteins formed in neurons?\nMinors\n• What are three oval symbols covering three or four variants (above the protein) in Fig. S1?\n• Line 639: two periods in the end of text.\n• Line 689-697: No comments on p.(Gln221Arg).\n• Line 708: p.(Thr210Met) should be p.(Thr207Met)?\n• Line 705: p.(Arg201Trp) instead of p.(Arg201Val).\n• Line 747: S1 is not analyzed in Fig. 6A.\n• Line 790: wer should be were.\n• Line 862: No D in Fig. S6.\n• Line 1300-1316: Fig.7 B and C become D and E in legend.\n• Figure 1: c.854G>A p.(Gly282Asp)) should be c.854G>A p.(Gly282Asp).\n• Only the first letter of the gene name of the model animals (fly and rat) are capitalized. All characters are capitals in Fig.3.\n• Possible multigenic origin could be commented regarding S11 as the similar finding was reported in PMID: 37600812 (if applicable) as the same authors are involved.\n• What are N50Rfs*7, L52P, M130I?, V362M and D394G in Figure S1?\n• This reviewer does not understand what gnomAD (1) and gnomAD (2) in Table 1 mean. please fill in the allele frequency of gnomAD as Table S1.\n• p.(Gly282Asp) is not shown in the 3D structural analysis figures.\n• In Fig.7 B, S12's father is a typo of S11's father.\n• In Fig.7 C, color-code or reshape the variants so that you can see the score of each variant. The right panel of Figure S5 B and the line graph below C should be similarly shown as indicated above."}, "author_response": "We would like to thank the referees for their insightful review of the manuscript and for their relevant comments, as well as yourself for processing this revision.\nIn the amended version, we addressed all the comments made, including additional experiments and more patients to improve the quality of the manuscript and to meet the expectations of the reviewers.\nTo address the reviewers’ remarks many new experiments in additional figures were required (new Figures: 2, 3D-J, 3M-N, 5C-D, 7A-B, 8C, 9, S1, S4, S5, S6A-B, S7D, S8, S11, S12) and led to substantial modifications of the text, figures and supplementary data. This includes additional multi-omics analyses and a human induced pluripotent stem cell (iPSC) model. In addition, six new cases were added compared to the previous version, which changed the numbering of affected subjects and variants. All redundancies across the tables and figures were removed, and we deleted Tables 1 and 2: (i) the variants are all reported in Fig. 1 and details regarding their nomenclature and pathogenicity are provided in Table S1 (in place of Table 1); whereas (ii) clinical findings are reported in Fig. 2 and detailed observations are listed in Table 2. We added a radar chart to indicate the main clinical features characterizing the disorder in Figure 2.\nThe new data indeed show impaired differentiation capacity of neural progenitor cells (NPGs) from ectodermal cells in our iPSC model, whereas differentiation into the 3 germ layers is less affected. These data explain a more central nervous system-directed manifestation of the disease. Additional multi-omics data indicate a critical involvement of PSMC5 in the maintenance of mitochondrial integrity and protein quality control, as well as support the dysregulated phospholipid metabolism also noted by lipidomics. Transcriptomics of iPSC-derived ectodermal cells revealed dysregulated differentiation signatures and altered cholesterol metabolism to support our major conclusions. Furthermore, these RNA sequencing experiments confirm a clear type I interferon signature in patient-derived T cells.\nOur point-by-point responses to the comments made are listed below.\nReviewer #1 (Remarks to the Author):\nGeneral remark: Review of paper by Kury et al. entitled Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies. In this manuscript the authors identified 23 unique variants in PSMC5 in 38 unrelated NDD individuals. The authors used a lot of different techniques and animal models (fly-mouse-rat) but to my opinion, these experiments were to superficial and not helping to elucidate the underlying general pathophysiology in the CNS.\nThe data obtained in the human T cells, on the other hand, is much more relevant and robust, but still do not fully explain the variety of clinical symptoms. I am missing the actual fundamental link between these mutations and the pathology.\n We thank the reviewer for the valuable comments and suggestions.\nWe agree that the clues provided in the previous version of the manuscript on the pathophysiological mechanisms leading from genotype to phenotype may have seemed insufficient. Consequently, we have designed additional experiments to provide further clues. On the one hand, (i) we have implemented an iPSC model. On the other hand, (ii) we have extended some of the cellular and animal models previously presented and (iii) included more multi-omics analyses.\n(i) iPSC model: We focused our assays on the c.973C>T p.(Arg325Trp) variant, as it is the most recurrent variant in the series, is unambiguously pathogenic according to functional explorations, and has a particularly marked multi-system action (cranio-facial system, heart, digestive system and ears) compared to the other variants (Fig. 2C). We assessed the pluripotency of the clones harboring this variant as well as their ability to differentiate into the three germ layers, including notably ectoderm, and into neural progenitor cells (NPC).\nIn brief, we found that the effects of the variant were initially well compensated by the iPSC, but became visible in later developmental stages. Thus, the pluripotency of the cell line was not significantly affected, as the iPSC could differentiate in the three germ layers (Fig. 9B; results: lines 1063-80), but the differentiation into neural progenitor cells was impaired (Fig. 9E-G; results: lines 1086-96). This problem of differentiation was already suggested by the under-expression of HES1 as a target of Notch signaling observed by transcriptomics of ectodermal cells harboring the variant (Fig. 9C; results: lines 1080-2). HES1 acts a transcriptional repressor and is known as a master regulator of neurogenesis. This observation alone establishes the genotype-phenotype correlation that was missing in the first version, showing that the developmental impact of the PSMC5 variant occurs during embryogenesis, at the neuronal progenitor stage (discussion: lines 1096-9).\n(ii) Rat hippocampal neurons: Complementary analyses showed that Psmc5 knockdown induced the aggregation of polyubiquitinated proteins, notably in the soma (Fig. 3M-N; results: lines 898-901). This finding should be considered in relation to the imbalance in the E/I ratio observed previously (discussion: lines 1127-38).\nDrosophila: In addition to the impairment of reversal learning previously highlighted, climbing was also negatively and very significantly impacted by rpt6 (PSMC5) knockdown (Fig. 3D-J; results: 883-9). Considering also the three possible cases of developmental regression (compared to milestones previously reached for language, cognition and or motor skills) noted in the cohort, this suggests that PSCM5 variants might induce premature aging or neurodegeneration (discussion: lines 1148-50).\nT cells from affected individuals: Transcriptomics and proteomics of more samples than in the previous version of the manuscript confirmed the dysregulation of type I interferon (IFN) signaling (Fig. 8C; results: lines 1035-6), and altered ribosome biogenesis (Fig S8A; results: lines 1007-11). Besides, the dysregulation of cholesterol metabolism highlighted by lipidomics on patient T cells was also suggested through deregulation of MSMO1 by iPSC-derived ectodermal cells harboring variant c.973C>T p.(Arg325Trp) (Fig. 9C; results: lines 1082-4). Exploring these pathophysiological avenues could be the topic of future studies.\nMajor remarks\nRemark 1: “Several mutations were “assumed de novo”. What does this mean exactly? Were parents tested yes or no? This is a very vague term to me.”\n The reviewer is right that the use of this term was not fully appropriate. It is officially used when a variant was found de novo but the biological relationship was not checked, which is automatically done when trio-exome or -genome sequencing is performed. We admit that we had misused the term here, by extending the notion to exceedingly rare variants not proven de novo by segregation analysis, but harbored by individuals whose biological parents were clinically unaffected. Consequently, in such situation, we corrected the mistake and simply stated that the variants were heterozygous and that parental samples were unavailable (Table S1).\nRemark 2: “Almost all patients carry a missense mutation. Patient S9 is homozygous for the mutation p.Thr207Met. This missense is also located in the AAA+ ATPase core domain, so one would expect that the heterozygous parents would also exhibit symptoms? Were they clinically examined, also for more subtle symptoms. It would also be interesting to take their T-cells along for the functional analyses.”\n We are aware that the presence of this unique homozygous variant could somehow be confusing. As a matter of fact, this observation does not challenge the mode of inheritance, which is autosomal dominant. Unfortunately, we are not allowed to disclose in the manuscript any information regarding the biological parents. In a confidential manner, we can inform the reviewers that the affected child (Subject 10 in the revised version of the manuscript) was born of incest between an uncle and his niece, and is now under the authority of another family member. It is not unlikely that the two parents have NDD, but we cannot prove it.\nA more convincing argument is the fact that we identified the same variant in an unrelated individual with NDD (Subject 11). Here again, segregation analysis could not be performed, and the clinical information provided is limited. Despite this lack of details, the report of this case shows that the variant in itself is associated with NDD at heterozygous state (see Table S2). This is also in line with our functional studies showing that the variant p.Thr207Met had a negative effect on neuritogenesis.\nRemark 3: Is there actually an average facial gestalt?\n We decided not to work on an average facial gestalt. Indeed, from our experience with other rare conditions, the series of individuals who provided photos is too small to generate significant results. The dysmorphic similarities between individuals are subtle and would be diluted in an average facial gestalt that would correspond to the face of a child with virtually no dysmorphic features. Therefore, we considered that it would be more informative to calculate the distance between the facial traits of the individuals compared to the large GestalMatcher database gathering thousands of photos from individuals with rare NDD.\nRemark 4: Given the dysregulation in type I IFN production, one would expect (auto)-immune problems. How thoroughly were the patients investigated for this?\n The reviewer is right. Clearly, none of the patients has been primarily linked to auto-immunity syndromes such as proteasome associated autoinflammtory syndromes (PRAAS). Yet, patients have not been specifically assessed for inflammatory or immune problems, and such problems were probably not emphasized by the parents who were mainly concerned by developmental issues. However, we realized that our description was probably not accurate enough, as many of the observations reported in Table 2 as ‘other clinical findings’ are consistent with symptoms seen in PRAAS and other autoinflammatory syndromes. Among them are lymphadenopathy, periodic fever in Subject 4 (S4); skin anomalies in S6; erythema and bilateral vascular congestion of lower extremities in S9; recurrent ear infections, chronic cough and frequent respiratory illness in S12; diabetes with arterial hypertension in S14; recurrent infections in S17; recurrent chest infections, chronic cough in S18; humoral immunodeficiency in S20 and S21; bone marrow failure, first presenting with congenital thrombocytopenia in S35; congenital thrombocytopenia (megakaryocytic) in S37; congenital thrombocytopaeina and anaemia in S38; iron deficiency anemia in S41; recurrent ear infections in S43.\nIn conclusion, there are links to autoimmunity from the clinical assessment of patients carrying PSMC5 variants and there is overlap with PRAAS. We therefore carefully discussed these overlaps in a relevant paragraph accordingly (lines 1165-73).\nRemark 5: Any problems in the carrier parents of patients S5 and S17? They should be investigated. The same holds true for the role of the proteasome in age-related neurodegenerative diseases. What is the age of the carrier mothers of patients S5 and S17? Were they investigated? Was segregation analysis done in these families? Maybe there are other family members that are older and carrier of the mutation.\n Patient S5’s mother (S6; aged 31 years), also harboring variant c.587del p.(Lys196Argfs*29), and patient S20 (formerly S17)’s mother (S21; aged 27 years) were enrolled in the study. Both were affected with symptoms similar to their children, confirming the pathogenicity of their respective variants, in line with the bioinformatics predictions and functional assessments. However, these two relatives are not old enough to provide any clues as to the possible involvement of proteasomal dysfunction in neurodegenerative diseases.\nNo further segregation analysis could unfortunately be performed in the two families, since because they did not respond to clinicians' invitations to investigate further.\n However, there is a body of evidence in favor of the involvement of proteasome dysfunction, related notably to PSMC5, in neurodegenerative diseases:\no PSCM5 deregulated expression would be a potential marker of the occurrence of Parkinson disease (PMID: 33042444)\no PSMC5 mRNA levels are significantly reduced in brain specimens from individuals with Parkinson’s disease dementia, dementia with Lewy bodies or Alzheimer’s disease (PMID: 28269775); these levels are associated with cognitive impairment in dementia with Lewy bodies.\no PSMC5 deregulation is included in a high risk AD signatures based on the APOE genotype (PMID: 20479757)\no Decreased PSMC5 mRNA levels could significantly predict the occurrence of Parkinson’s disease (PMID 33042444).\nRemark 6: Do proteasome-linked genes, and PSMC5 in particular, pop-up in GWAS studies?\n Numerous associations have been demonstrated between proteasomal gene variants and a wide variety of traits. If we limit ourselves to the genes we know best:\n PSMD12 variants have been associated essentially with body mass index (https://www.ebi.ac.uk/gwas/genes/PSMD12);\n PSMD11 variants have been associated with systolic blood pressure, but also to ADHD and schizophrenia (https://www.ebi.ac.uk/gwas/genes/PSMD11);\n PSMC3 variants emerged from more GWAS, including notable studies dealing with depressive symptoms, neurocitism, cognitive function measurement, intelligence and Alzheimer (https://www.ebi.ac.uk/gwas/genes/PSMC3)\n PSMC5 variant were highlighted in six studies, including one dealing with IGF-1 measurement and another one with memory decline in normal cognition (https://www.ebi.ac.uk/gwas/genes/PSMC5). This last association (PMID: 37984853) is in line with our observations in Drosophila discussed in the manuscript.\n Beyond GWAS, the 26S proteasome and certain other proteasome genes have been identified as hub genes involved in early onset schizophrenia in a recent preprint (https://doi.org/10.21203/rs.3.rs-5833160/v1).\nFurthermore, a large exome-based international study on developmental disorders point to a significant enrichment of de novo rare PSMC5 variants–predicted pathogenic–in the cohort (PMID: 33057194), which is consistent with our observations. This is indicated in the manuscript (discussion: lines 1095-8)\nPSMC5 was also proposed as a prognostic biomarker for the survival to numerous cancerous disorders, including hepatocellular carcinoma survival (PMID: 38370535), lung adenocarcinoma (PMID: 35647031), glioma (PMID: 34804978) or breast-cancer (PMID: 34329194). PSMC5 would also participate in the pathophysiological mechanism of COVID-19 and would thus represent a potential therapeutic target (PMID: 34004362).\nAside from these links with malignancies, studies of particular interest highlight the involvement of PSMC5 in the pathogenesis of neurological diseases, as it would for instance drive the trinucleotide expansions involved in disorders such as Huntington’s disease (PMID: 23620289). Furthermore, its mRNA levels are decreased in blood samples from children with cerebral malaria (PMID: 28934429).\nWe did not mention the aforementioned articles in the manuscript, as they did not directly relate to the disorder we describe or to its clinical course. On the other hand, we cited three studies proposing PSMC5 as potential biomarker (main text; lines 1186-8, see above). In more details:\no PSCM5 deregulated expression would be a potential marker of the occurrence of Parkinson disease (PMID: 33042444)\no PSMC5 mRNA levels are significantly reduced in brain specimens from individuals with Parkinson’s disease dementia, dementia with Lewy bodies or Alzheimer’s disease (PMID: 28269775); these levels are associated with cognitive impairment in dementia with Lewy bodies.\no PSMC5 deregulation is included in a high risk AD signatures based on the APOE genotype (PMID: 20479757)\nRemark 7-9: Minor remarks\n Remark 7: L 268: delete second “in this context”\n It is done (lines 300-1).\n Remark 8: L 653: day one of development? To what exact developmental time point does this correspond?\n We suppose that this term may have been confusing. Actually, it corresponds to day in vitro 1 (DIV1), which we used as a replacement of “day one of development” in the revised version of the manuscript (line 893).\nHere, rat hippocampal neurons were purchased from Innoprot. They were isolated from embryonic day 18 rat brain hippocampus and were plated onto a substrate at DIV0, before lenti-virus infection at DIV1.\nWith the term “development” initially used, we intended to reflect early differentiation, comparable to the in vivo development of neurons transitioning from late embryonic to early postnatal stages.\n Remark 9: In the abstract, the authors state that they looked in “human” hippocampal neurons? Typo probably?\n We apologize for the mistake. It was corrected to “mouse” (line 249).\nReviewer #2 (Remarks to the Author):\nAuthors describe a total of 23 pathogenic PSMC5 variants in 38 individuals of neurodevelopmental disorders with various other organs involvement. PSMC5 encodes the AAA-ATPase proteasome subunit PSMC5/Rpt6, a component of the 26S proteasome complex. Several multimodal functional analyses have been performed: PSMC5 overexpression resulting in altered human hippocampal neuronal morphology, PSMC5 knockdown causing impaired reversal learning in Drosophila and loss of excitatory synapses in rat hippocampal neurons, abnormal proteasome assembly defects associated with most PSMC5 variants, abnormalities of innate immune signaling, mitophagy and lipid metabolism together with their witness of PKR and GCN2 kinases potentially ameliorating immune dysregulations in affected patients. This reviewer thinks identification of 23 novel variants and various functional assays are well-done, providing new scientific aspects in the new proteasomopathy, and suggest several points which may improve the manuscript. Recently, neurodevelopmental proteasomopathies with abnormalities of PSMD12, PSMC3, and PSMB1 have been reported, and PSMC5 abnormalities were added by this manuscript.\nWe thank the reviewer for the valuable comments and suggestions.\nMajors\nRemark 1: Since the impact of each variant is complex and varies depending on the target of observation (e.g., effects on PSMC5 stability, proteasome assembly, mitophagy, IFN signature, etc), it is easier to understand if functional impacts by pathogenic variants are summarized in a figure or table.\n This is an excellent suggestion. The findings are now summarized in Table S1.\nRemark 2: A scheme summarizing the causative genes and phenotypes of proteasome-related neurodevelopmental proteasomopathies and diseases with similar cellular dynamics to this disease, such as CANDLE and PRAAS, would be helpful for the understanding of readers.\n Thank you for the suggestion, all known proteasome gene variants and phenotypes summarized in the new Fig. S1. In addition, a review published recently summarizes all known proteasome gene variants and the clinical complexity of these proteasomopathies (PMID: 39220754). It is now cited in the manuscript (lines 291 and 1167).\nRemark 3: Are there any phenotype-genotype correlation among the position of variants regarding 3D structure, functional domains, in vitro phenotypes?\n The proposed analysis is particularly relevant, considering the size of the case series presented. While preparing the earlier version of the manuscript, we had explored potential genotype-phenotype correlations but had not identified any significant findings. However, during the revision of Figure 1, some trends have emerged that are worth noting. It appears that cardiovascular (conotruncal malformations) and musculoskeletal signs are more particularly prominent in association with the p.Arg325Trp variant and the splice variant c.970-2A>G predicted to induce skipping of the exon 10 coding the region containing the p.Arg325Trp variant. Besides, all the patients harboring this variant also have feeding difficulties, and several of them have hearing loss, two being sensorineural. By contrast, we noted that patients with variants affecting the first part of the protein frequently presented with short stature.\nWe mentioned these observations in the results section (results: lines 860-9).\nRemark 4: Two affected mothers of S5 and S17 should be characterized at the similar level to their children. Perhaps include such detailed information in Tables 1, 2 and S2.\n The reviewer is right. Both were affected with symptoms similar to their children, confirming the pathogenicity of their respective variants, in line with the bioinformatics predictions and functional assessments. Please, see also our answer to reviewer 1’s remark 5.\nRemark 5: Parents of S9 with a homozygous variant are assumed to have the variant heterozygously. Even if DNA is unavailable, their phenotypic information should be helpful to consider how the homozygous variant should be positioned in this manuscript. Please comment on the phenotypic difference between heterozygosity and homozygosity. Is this family consanguineous?\n We fully agree and tried as much as possible to get information, but it was impossible. Unfortunately, we are not allowed to disclose in the manuscript any information regarding the biological parents. In a confidential manner, we can inform the reviewers that the affected child (now Subject 10 in the revised version of the manuscript) was born of incest between an uncle and his niece, and is now under the authority of another family member. It is not unlikely that the two parents have NDD, but we cannot prove it.\nOn the other hand, we included an additional patient heterozygous for the very same variant. Please, see our answer to reviewer 1’s remark 2.\nRemark 6: Facial image analysis using GestaltMatcher is interesting. Please comment of the most typical face of PSMC5 variants if possible. S9 is a homozygous patient and others are all heterozygous patients, though.\n Although most of the patients have dysmorphic facial features, these ones are very heterogeneous. It is therefore very difficult to draw a typical face of a patient affected by the PSMC5-related NDD. As discussed for reviewer 1’s remark 3, we are not convinced by the interest of an average facial gestalt, considering the small number of patients contributing to facial analysis. The similarities between the patients clearly emerge when calculating the distance separating the patient with PSMC5 patients from others, but this method takes into account subtle traits that would not appear on an average portrait.\nRemark 7: S26 with p.(Pro320His) has a twin with similar presentations and ADHD in the father. How about their genetic inheritance? The allele count for gnomaAD also has 8 alleles.\n We agree that this information would be invaluable. Unfortunately, the family did not reply to our request for a segregation analysis. While we will continue our efforts, it is unlikely that we will obtain any results of segregation analysis in time. On the other hand, bioinformatic predictions and molecular data strongly support the pathogenicity of this variant. Although its relative frequency does not provide a strong argument for pathogenicity, we have accounted for it in our predictions. Moreover, gnomAD v4 includes individuals who may have neurological or neuropsychiatric conditions (e.g., bipolar disorder, schizophrenia, depression), which could potentially be linked to the PSMC5 variant. When excluding the UK Biobank data and analyzing gnomAD version v4.1.0 (non-UKB), only two individuals heterozygous for c.959C>A p.(Pro320His) are identified. Therefore, we do not consider this frequency to be a sufficient reason to dismiss the pathogenicity of the p.(Pro320His) variant. However, the relative frequency does suggest that its effects are likely less severe than those of the recurrent c.959C>G p.(Pro320Arg) variant, which affects the same nucleotide. This conclusion aligns with our clinical and functional observations.\nRemark 8: Different results between SHSY-5Y cell experiments and patients’ T cells was considered to be due to compensatory effects. But this could be due to difference cell natures.\n We thank the referee for raising this valid point and appreciate the opportunity to elaborate on this aspect. The contrasting protein levels of the p.(Arg201Trp), p.(Glu250Val), p.(Glu250Val), and p.(Arg325Trp) PSMC5 variants observed between SH-SY5Y cells and T cells may have several explanations. As mentioned in the original manuscript, one possibility is that T cells exhibit unbalanced allele expressionwith the wild-type allele being expressed at higher levels than the mutant counterpart to compensate for PSMC5 deficiency. In addition, as suggested by the referee, these differences could arise from the inherent characteristics of the two cell types or even from differences in experimental setups. Unlike T cells which endogenously express the variants, SH-SY5Y cells were transfected to ectopically express them from a plasmid-driven viral promoter at very high levels. Overexpression of individual proteasome subunits can disrupt the stoichiometric balance, rendering them more prone to degradation (PMID: 34382105). Thus, it is plausible that the SH-SY5Y system is more sensitive to detecting subtle effects of the variants on proteasome subunit turnover compared to T cells. This point has now been clarified and discussed in more detail in the revised version of the manuscript (results; lines 966-8).\nRemark 9: Increased LMNA expression may cause premature aging. How about the accumulation of falnesylated pre-laminA (progerin)?\n Since the previous version of the manuscript, we performed additional proteomic analyses on a larger number of individuals than in the first series of proteomic analyses. The results obtained are therefore more robust. In this case, we were unable to reproduce the increase in LMNA levels as an event shared by all the patients tested. Consequently, we did not further investigate the possibility of farnesylated pre-lamin A accumulation.\nEven if LMNA is eventually not deregulated in all patients, other findings in the second proteomics series suggest that the hypothesis of premature aging or neurodegeneration caused by PSMC5 dysfunction remains worth exploring. In PSMC5 patients, we noted for example increased expression levels of clusterin (CLU), which was proposed as a potential biomarker for Alzheimer’s disease (PMID: 38017342). Yet, we are aware that such observations are fragile and would need to be replicated in more patients. For this reason, we have remained cautious in not mentioning them here, and instead focusing on the most significant results observed in the omics analyses.\nRemark 10: Why do patients with PSMC5 variants show no obvious signs of autoinflammation?\n This comment is quite similar to reviewer 1's remark 4 about (auto)immune problems. We realized that our words may have sounded misleading. As a matter of fact, patients with PSMC5 variants do not show the striking autoinflammatory symptoms that are the hallmark of PRAAS. However, When the clinical information provided by clinicians is dissected, many of the symptoms present in multiple patients in the series are suggestive of, or compatible with, immune problems (Table S1). These symptoms are detailed in the answer to reviewer 1's remark 4. Besides, we modified the related paragraph in the discussion (lines 1167-70).\nA careful clinical re-assessment of all the patients focused on autoimmunity and immune dysregulation would be relevant, but not feasible in time. We are however convinced that the publication of this study will raise awareness that proteasomopathies are linked to type I interferon dysregulation and autoimmunity.\nRemark 11: Are there any neurological symptoms that appear in late onset due to accumulation of ubiquitinated proteins in older patients?\n Very few adult patients are included in the cohort, and the observations are thus limited. However, brain MRI showed cerebral atrophy or a progression of brain lesions in three young adults, which could reflect neurodegeneration and strengthen the importance of clinical follow-up to detect possible signs of premature neurodegeneration (discussion; lines: 1150-4).\nBesides, in other neurodevelopmental proteasomopathies that we investigate in parallel, we are aware of cases of bipolar disorder or schizophrenia, hence our answer above regarding variant p.(Pro320His). We have not found yet any case of Alzheimer’s or Parkinson’s disease, but the few adults included are still relatively young. The cohort presented in this study on PSMC5 is therefore not necessarily the most suitable for studying this type of disease progression. Only the cases of regression, in particular those mentioned in the manuscript, support the idea of possible neurodegenerative mechanisms. Here again, further specific studies will be needed to validate our hypotheses.\nRemark 12: Since PSMC5 function may differ depending on the time of development and tissues, it is preferable to evaluate the pathophysiology using not only cultured cells but also knock-in mice.\n We fully agree with the reviewer. However, there is no knock-in mouse model available, and it would have taken too much time to generate and implement it. To avoid revision delays, we implemented an iPSC model, focusing on the c.973C>T p.(Arg325Trp) variant, as explained at the beginning of the letter (see our answer to reviewer 1’ general remark). We informed the editor about this choice of model.\nRemark 13: Are inclusion bodies composed of ubiquitinated abnormal proteins formed in neurons?\n Yes, indeed. By immunostaining using anti-ubiquitin antibodies, we showed an accumulation of ubiquitinated proteins in Psmc5-KD hippocampal neurons (Fig. 3M,N; lines 898-901).\nMinors\nRemark 14: What are three oval symbols covering three or four variants (above the protein) in Fig. S1?\n We used these cloud-like symbols to highlight clusters of variants. We specified it in the caption of Fig. S2 (formerly Fig. S1).\nRemark 15: Line 639: two periods in the end of text.\n It was deleted.\nRemark 16: Line 689-697: No comments on p.(Gln221Arg).\n The reviewer is right. We indicated that the steady-state protein expression levels of PSMC5/Rpt6 were mildly reduced by variant p.(Gln221Arg). In addition, we revised some of our interpretations for other variants compared to the previous version of the manuscript (results: lines 932-8).\nRemark 17: Line 708: p.(Thr210Met) should be p.(Thr207Met)?\n The error of nomenclature is now corrected (results: line 952).\nRemark 18: Line 705: p.(Arg201Trp) instead of p.(Arg201Val).\n It is corrected (results: line 949).\nRemark 19: Line 747: S1 is not analyzed in Fig. 6A.\n We modified the text to distinguish between Fig. 6A and Fig. 6B -where S1 was included in the analyses (results: lines 989-94).\nRemark 20: Line 790: wer should be were.\n The related sentence has been slightly modified, resulting in the disappearance of this typo.\nRemark 21: Line 862: No D in Fig. S6.\n This mention originally appeared in the discussion, which has been considerably modified in the current version of the manuscript. Therefore, it no longer exists. The new paragraph related to the disruption of lipid homeostasis lies between lines 1183 and 1187.\nRemark 22: Line 1300-1316: Fig.7 B and C become D and E in legend.\n The numbering was indeed not correct. In the present version, Figure 7 became Figure 8, and changes were made between the panels that compose it. We checked that the legend was consistent with the figure (lines 1725-57)\nRemark 23: Figure 1: c.854G>A p.(Gly282Asp)) should be c.854G>A p.(Gly282Asp).\n The supplementary bracket was removed.\nRemark 24: Only the first letter of the gene name of the model animals (fly and rat) are capitalized. All characters are capitals in Fig.3.\n The nomenclature of genes and proteins was modified in Fig. 3 to comply with the official rules for each organism: (i) for Drosophila melanogaster, gene in lower case and italics; (i) for Ratus norvegicus, gene italicized beginning with an uppercase letter followed by all lowercase letters, and protein symbols using all uppercase letters not italicized.\nRemark 25: Possible multigenic origin could be commented regarding S11 as the similar finding was reported in PMID: 37600812 (if applicable) as the same authors are involved.\n This is indeed a possible mechanism that we evoked in the results section, where we report this specific case (lines 978-80). The reference suggested by the reviewer is especially relevant, because one of the cases presented involved a potential trigenic mechanism, with the contribution of a PSMC5 splice site variant.\nRemark 26: What are N50Rfs*7, L52P, M130I?, V362M and D394G in Figure S1?\n We thank the reviewer for noting these errors. These variants escaped our attention when we cleansed the data from the various figures in the manuscript. They had been included in first place, as they had been found in patients whose families eventually did not want to participate in the study. We corrected the figure, so that only the variants included appear.\nRemark 27: This reviewer does not understand what gnomAD (1) and gnomAD (2) in Table 1 mean. please fill in the allele frequency of gnomAD as Table S1.\n As mentioned above, we deleted Table 1 to avoid redundancies with Figure 1 and Table S1. The allele frequencies from gnomAD are indicated in Table S1.\nRemark 28: p.(Gly282Asp) is not shown in the 3D structural analysis figures.\n The variant was added to the figure.\nRemark 29: In Fig.7 B, S12's father is a typo of S11's father.\n We corrected the labels in Fig. 8C (formerly Fig. 7B). Due to the change in numbering, “S11’s father” is now “S14’s father”.\nRemark 30: In Fig.7 C, color-code or reshape the variants so that you can see the score of each variant. The right panel of Figure S5 B and the line graph below C should be similarly shown as indicated above.\n We modified the color code in Fig. 8C-D (formerly Fig. 7B-C). We used a specific color for each patient and for S14’s father who harbors the PSMD11 variant p.(Arg90*).\n We also modified Fig. S6A-B (formerly S5A-B) so that the fold change of PSMC5 levels can be detailed for each variant."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Re-review of paper by Kury et al.\nThe authors have addressed my comments and also included additional data using a human iPSC model. These additional experiments have certainly improved the quality and content of the paper by providing more insight into the underlying pathophysiology.\nExcept for some minor typos, I have no additional questions.", "reviewer_2": "Authors added new experiments such as additional multi-omics analyses, iPSC model experiment, and recruited six new PSMC5-aberrant cases. Figures and Tables are newly structured. The iPSC experiment disclosed the impairment differentiation of neural progenitor cells, and additional mult-iomics experiments showed abnormal mitochondrial integrity and protein quality control and dysregulated cholesterol metabolisms. Authors’ efforts are significant. Though many data were statistically analyzed and sometimes showed subtle change, this reviewer recognizes authors are reasonably figure out neurodevelopmental proteosomopathies by PSMC5 aberration as shown below in response to this reviewer’s previous concerns.\nMajors\nRemark 1: Table S1 is easy to understand the functional impact of variants.\nRemark 2: Fig S1 effectively illustrates which and how variants of subunit genes of 26 proteasome lead to human disorders.\nRemark 3: Very good to know that p.Arg325Trp and c.970-2A>G leading to exon 10 skipping containing Arg325 cause cardiovascular and musculoskeletal signs.\nRemark 4: Two affected mothers are confirmed but unfortunately not described in detail because of their no-cooperation.\nRemark 5: The complicated situation is understood by this reviewer.\nRemark 6: This reviewer agrees that it is difficult to show an average portrait or a typical face.\nRemark 7: Authors’ consideration of p.(Pro320His) seems reasonable which can be described in the manuscript.\nRemark 8: It is good to describe this reviewer’s concern about different results came from different cells used.\nRemark 9: This reviewer agrees with no mention of falnesylation of progerin as it was not reproducible.\nRemark 10: As this reviewer expected, autoinflammation is likely involved and mentioned as the response to Remark 4 by the Reviewer 1.\nRemark 11: Possible premature neurodegeneration is very interesting but exceptional and mentioned in the revised manuscript.\nRemark 12: Instead of knock-in mouse, authors implemented an iPSC model to address c.973C>T p.(Arg325Trp).\nRemark 13: OK.\nMinors\nRemark 16: Commented on p.(Gln221Arg).\nOther minor remarks are well responded.\nNew other Errors and typos in the revised manuscript.\nPage 6, line 247: Its. Knockdown>Its knockdown\nPage 27, line 807: (7/44 subjects)>(6/44 subjects) and (8/44)>(9/44) based on the new Figure 1A\nFigure 1B: Arg325Trp 8>9 and Pro320His 7>6 based on the new Figure 1A\nPage 27, line 817-818: the American College of Medical Genetics and Genomics (ACMG) guidelines> the American College of Medical Genetics and Genomics/the Association for Molecular Pathology (ACMG/AMP) guidelines\nPage 27, line 821: Fig. 1A>Fig. 2A\nPage 32, line 952: (p.Arg325Trp)>p.(Arg325Trp)\nFigure 5C: S23 (p.Pro320Arg)>S25 (p.Pro320Arg)\nFigure 6A: S7 (V7)>S7 (V6)\nFigure 7B: gene name is hard to see. This reviewer suggests a bit enlarged version is recommended.\nFigure 7E legend: six NDD subjects, but this reviewer sees 7 red points in Figure 7E."}, "author_response": "We would like to thank once again the referees for their thorough review and for recommending the manuscript for publication to the editor. Since Reviewer #1 was satisfied with all the changes made in the prior version, we detailed below only the remaining minor comments made by Reviewer #2.\nOur point-by-point responses to the comments made are listed below.\nReviewer #2:\nPage 6, line 247: Its. Knockdown>Its knockdown\n We thank the reviewer for spotting this typo, which has been removed from the manuscript as the sentence has been rewritten.\nPage 27, line 807: (7/44 subjects)>(6/44 subjects) and (8/44)>(9/44) based on the new Figure 1A\n These figures had indeed not been updated as they should have been. This has now been done.\nFigure 1B: Arg325Trp 8>9 and Pro320His 7>6 based on the new Figure 1A\n As with the previous comment, we have corrected this error too.\nPage 27, line 817-818: the American College of Medical Genetics and Genomics (ACMG) guidelines> the American College of Medical Genetics and Genomics/the Association for Molecular Pathology (ACMG/AMP) guidelines\n We have followed this suggestion and indicated the full name for the ACMG/AMP.\nPage 27, line 821: Fig. 1A>Fig. 2A\n We have corrected this error.\nPage 32, line 952: (p.Arg325Trp)>p.(Arg325Trp)\n We moved the bracket to the right place.\nFigure 5C: S23 (p.Pro320Arg)>S25 (p.Pro320Arg)\n We have modified the label in the figure.\nFigure 6A: S7 (V7)>S7 (V6)\n We have modified this label too.\nFigure 7B: gene name is hard to see. This reviewer suggests a bit enlarged version is recommended.\n We have redrawn the figure, in order to use larger font sizes.\nFigure 7E legend: six NDD subjects, but this reviewer sees 7 red points in Figure 7E.\n We are grateful to the reviewer for noticing this discrepancy. We had indeed forgotten to indicate one patient in the legend. We have now listed six patients: S1, S7, S14, S15, S25, S36, and S37."}]

Decision letter