# Understanding neurodevelopmental proteasomopathies as new rare disease entities: A review of current concepts, molecular biomarkers, and perspectives

Silvestre Cuinat, Stéphane Bézieau, Wallid Deb, Sandra Mercier, Virginie Vignard, Bertrand Isidor, Sébastien Küry, Frédéric Ebstein

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Silvestre Cuinat, Stéphane Bézieau, Wallid Deb, Sandra Mercier, Virginie Vignard, et al. Understanding neurodevelopmental proteasomopathies as new rare disease entities: A review of current concepts, molecular biomarkers, and perspectives. Genes & Diseases, 2023, pp.101130. 10.1016/j.gendis.2023.101130. hal-04264182

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Understanding neurodevelopmental proteasomopathies as new rare disease entities:
A review of current concepts, molecular biomarkers, and perspectives

Silvestre Cuinat, Stéphane Bézieau, Wallid Deb, Sandra Mercier, Virginie Vignard,
Bertrand Isidor, Sébastien Küry, Frédéric Ebstein

<figure><img src="image_3.png" /></figure>

PII: S2352-3042(23)00413-0

DOI: https://doi.org/10.1016/j.gendis.2023.101130

Reference: GENDIS 101130

To appear in: *Genes & Diseases*

Received Date: 15 May 2023

Revised Date: 30 July 2023

Accepted Date: 19 August 2023

Please cite this article as: Cuinat S, Bézieau S, Deb W, Mercier S, Vignard V, Isidor B, Küry S, Ebstein F, Understanding neurodevelopmental proteasomopathies as new rare disease entities: A review of current concepts, molecular biomarkers, and perspectives, *Genes & Diseases*, https://doi.org/10.1016/j.gendis.2023.101130.

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# Understanding neurodevelopmental proteasomopathies as new rare disease entities: A review of current concepts, molecular biomarkers, and perspectives

Silvestre Cuinat<sup>a,b</sup>, Stéphane Bézieau<sup>a,b</sup>, Wallid Deb<sup>a,b</sup>, Sandra Mercier<sup>a,b</sup>, Virginie Vignard<sup>a,b</sup>, Bertrand Isidor<sup>a,b</sup>, Sébastien Küry<sup>a,b</sup>, Frédéric Ebstein<sup>b,*</sup>

<sup>a</sup> Nantes Université, CHU Nantes, Service de Génétique Médicale, Nantes F-44000, France

<sup>b</sup> Nantes Université, CHU Nantes, CNRS, INSERM, l'institut du thorax, Nantes F-44000, France

E-mail address: frederic.ebstein@univ-nantes.fr (F. Ebstein).

Peer review under responsibility of Chongqing Medical University.

## Abstract

The recent advances in high throughput sequencing technology have drastically changed the practice of medical diagnosis, allowing for rapid identification of hundreds of genes causing human diseases. This unprecedented progress has made clear that most forms of intellectual disability that affect more than 3% of individuals worldwide are monogenic diseases.

Strikingly, a substantial fraction of the dominant forms of intellectual disability is associated with genes related to the ubiquitin-proteasome system, a highly conserved pathway made up of approximately 1200 genes involved in the regulation of protein homeostasis. This group is currently emerging a new class of neurodevelopmental disorders specifically caused by proteasome pathogenic variants which we propose to designate "neurodevelopmental"

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<footnote>* Corresponding author.</footnote>
proteasomopathies". Besides cognitive impairment, these diseases are typically associated
with a series of syndromic clinical manifestations, among which facial dysmorphism, motor
delay, and failure to thrive are the most prominent ones. While recent efforts have been made
to uncover the effects exerted by proteasome variants on cell and tissue landscapes, the
molecular pathogenesis of neurodevelopmental proteasomopathies remains ill-defined. In this
review, we discuss the cellular changes typically induced by genomic alterations in
proteasome genes and explore their relevance as biomarkers for the diagnosis, management,
and potential treatment of these new rare disease entities.

**Keywords:** Biomarkers; Loss-of-function variants; Neurodevelopmental disorders;
Proteasome; Rare diseases; Therapeutic targets

37

**Introduction**

38

Intellectual disability is a neurodevelopmental disorder (NDD) occurring before adulthood which affects up to 3 % of the worldwide population and is typified by both cognitive impairment and deficits in adaptive behavior ¹⁻³. Approximately two-thirds of intellectual disability forms have a genetic cause with a predominance of monogenic inheritance ⁴.

Thanks to the recent implementation of high-throughput sequencing technology in clinical care, the number of new genes associated with intellectual disability is growing steadily.

Nevertheless, in 2021, Kaplanis and colleagues suggested that about 1000 genes causing NDD have not been fully identified yet ⁵. Many — if not all — of the genes associated with NDD are involved in basic biological processes, including those regulated by the ubiquitin-proteasome system (UPS) ⁶. With more than 1200 genes ⁷, the UPS is a highly conserved pathway across eukaryotic species that preserves protein homeostasis by targeting ubiquitin-
tagged proteins to degradation by the proteasome ⁸. Ubiquitin is a 76-amino acid peptide that covalently modifies protein substrates destined to be removed via the sequential action of three different enzymes, namely E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases ⁹. Ubiquitin itself may be subjected to ubiquitination, thereby allowing the formation of polyubiquitin chains on protein substrates. It is widely accepted that proteasomes predominantly recognize polyubiquitin chains in which ubiquitin moieties are joined together via Lys48-linkages ⁹. Ubiquitin-modified proteins are typically degraded by the 26S proteasome, a giant protein complex composed of a 20S core particle (CP) capped at one end by a 19S regulatory particle (RP) ¹⁰ (Fig. 1). The 19S RP can be further divided into two parts, namely the lid and the base¹⁰. As depicted in Figure 1, the lid is made up of eight scaffolding subunits — PSMD3/Rpn3, PSMD12/Rpn5, PSMD11/Rpn6, PSMD6/Rpn7, PSMD7/Rpn8, PSMD13/Rpn9, PSMD8/Rpn12, and SEM1/Rpn15 — and one deubiquitinating enzyme PSMD14/Rpn11 ¹⁰. As for the base of the 19S RP, it is formed by the association of six different AAA+ ATPase subunits (Rpt1-6 encoded by PSMC1-6 genes) with the four non-ATPase subunits PSMD2/Rpn1, PSMD1/Rpn2, PSMD4/Rpn10, and ADRM1/Rpn13 ¹⁰ (Fig. 1). In contrast to the lid whose subunits may self-assemble, the biogenesis of the base relies on four assembly chaperones, namely PSMD9/p27, PSMD10/p28, PSMD5/S5b, and PAAF1/Rpn14 ¹¹. The base and the lid then associate following their individual maturation ¹¹. One major function of the 19S RP consists of sensing ubiquitin-modified proteins via the PSMD4/Rpn10 and ADRM1/Rpn13 subunits which serve as ubiquitin receptors. Following recognition and binding, protein substrates are then deubiquitinated by PSMD14/Rpn11, USP14, and UCHL5 and finally unfolded by the six AAA+ ATPase PSMC1-6/Rpt1-6 before translocation into the 20S CP ¹².

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As illustrated in Figure 1, the 20S CP is built by the juxtaposition of four heptameric rings
consisting of two outer α-rings and two inner β-rings each composed of seven different α- and
β-subunits encoded by the *PSMA1-7* and *PSMB1-7* genes, respectively <sup>10</sup>. The 20S CP arises
from the dimerization of 16S α/β heterodimer precursor complexes which themselves
assemble with the assistance of the four chaperones *PAC1-4* encoded by the *PSMG1-4* genes
and the proteasome maturation protein *POMP* encoded by *POMP* <sup>11</sup>. The 20S CP ensures the
degradation of translocated protein substrates into short peptides via the catalytic β-subunits
(*PSMB6/β1*, *PSMB7/β2*, *PSMB5/β5* carrying caspase-like, trypsin-like, and chymotrypsin-like
activities, respectively) <sup>13</sup>. Depending on the composition of the β-ring and/or association with
other RP than 19S, several proteasome isoforms may arise. These include notably
immunoproteasomes in which the *PSMB6/β1*, *PSMB7/β2*, and *PSMB5/β5* standard subunits
have been replaced with the *PSMB9/β1i*, *PSMB10/β2i*, and *PSMB8/β5i* inducible ones <sup>14</sup> (Fig.
1). While immunoproteasomes are constitutively expressed in immune cells, their biogenesis
can be induced in non-immune cells following exposure to inflammatory stimuli such as
interferons (IFN)<sup>15</sup>. It is understood that immunoproteasomes degrade protein substrates faster
than their standard counterparts, a property that makes them critical regulators of protein
homeostasis and MHC class I antigen presentation <sup>16,17</sup>. As depicted in Figure 1, the 20S CP
of proteasomes may contain other atypical subunits, namely *PSMA8/α4s* and *PSMB11/β5t*,
which are exclusively found in the testis and thymus, respectively <sup>18,19</sup>. *PSMA8/α4s* is a
crucial component of the spermatoproteasome, responsible for the breakdown of meiotic
proteins like *RAD51* and *RAP1*, a process essential for the progression of meiosis during
spermatogenesis <sup>20</sup>. On the other hand, *PSMB11/β5t* is a part of the thymoproteasome, and its
role involves providing MHC class I-restricted peptides, which facilitates the positive
selection of CD8<sup>+</sup> T cells <sup>21</sup>, even though the precise mechanisms underlying this process
remain poorly understood.
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101 Independently of the α- and β-subunits, several additional particles other than the 19S RP may

102 bind to the 20S CP to form extra proteasome types. For instance, as illustrated in Figure 1,

103 most 20S complexes in the cell are associated with a PA28-αβ ring made up of three

104 PSME1/PA28-α- and four PSME2/PA28-β subunits<sup>22</sup> or four PSME1/PA28-α- and three

105 PSME2/PA28-β subunits<sup>23</sup> to build PA28-capped proteasomes. It has been shown that PA28-

106 20S complexes are capable of degrading protein substrates such as oxidized proteins in an

107 ATP- and ubiquitin-independent fashion<sup>24</sup>. Apart from PA28-αβ, proteasomes may also

108 associate with PSME3/PA28γ or PSME4/PA200 (Fig. 1) to modulate the turnover of nuclear

109 proteins, in particular cell cycle regulators and histones, respectively<sup>25,26</sup>. Less prominent

110 regulators include ECM29 and PI31 whose binding to proteasomes may exert various

111 functional consequences such as 26S complex disassembly, redistribution, and inhibition<sup>27,28</sup>.

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113 Genetics of proteasomopathies

114 The term "proteasomopathies" is a recent portmanteau used to design disorders caused by
115 proteasome dysfunction <sup>29</sup>. Proteasomopathies thus represent a rather large group of
116 heterogenous diseases which, depending on their molecular etiology, may be further classified
117 into different subtypes. In this review, we will focus on primary proteasomopathies, namely
118 on congenital defects resulting from *de novo* and/or inherited variants affecting at least one of
119 the 52 genes encoding proteasome subunits, assembly factors, and/or regulators. These
120 diseases are rare or even ultra-rare with so far less than 120 cases reported worldwide. This
121 rarity is in sharp contrast to secondary proteasomopathies, which include well-known late-
122 onset idiopathic proteasome deficiencies such as neurodegenerative diseases whose genetic
123 component is much less well-defined.

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As mentioned above, primary proteasomopathies are early-onset disorders caused by genomic alterations in proteasome genes. Intriguingly, proteasome lesions convey two distinct clinical phenotypes characterized by either systemic autoinflammation or neurodevelopmental delay.

Although these manifestations are not strictly mutually exclusive, they are considered to underlie two distinct disease entities which we will refer to as (i) autoinflammatory and (ii) neurodevelopmental proteasomopathies. To date, the molecular basis for the dual phenotype is not understood. A peculiarity that is even harder to follow given that these two phenotypes cannot be mapped to specific proteasome variants and/or genes.

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## Autoinflammatory proteasomopathies (CANDLE/PRAAS)

From a chronological point of view, autoinflammation was the first clinical manifestation associated with proteasome loss-of-function. In 2010, Agarwal et al reported the first p.(Thr75Met) homozygous missense variant in the *PSMB8* proteasome gene ³⁰ in patients suffering from an autoinflammatory syndrome named joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy syndrome ³¹. To the best of our knowledge, approximately 36 variants have been identified in seven proteasome genes in 38 individuals, all presenting with typical signs of systemic autoinflammation (Table 1).

Meanwhile, two different names have been given to these conditions, namely chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) and proteasome-associated autoinflammatory syndromes (PRAAS) ³². Strikingly, genes frequently altered in CANDLE/PRAAS include the three genes coding for the inducible subunits PSMB8/β5i, PSMB9/β1i, and PSMB10/β2i, suggesting that these diseases were specifically caused by immunoproteasome loss-of-function. It became, however, rapidly evident that CANDLE/PRAAS were not exclusively associated with immunoproteasome genes and that lesions in other α- and/or β-subunits or even proteasome assembly factors (POMP, PSMG2)
could trigger the disease as well (Table 1). Remarkably, as shown in Figure 2, all proteasome subunits affected in CANDLE/PRAAS are localized within the 20S CP, suggesting that the disease selectively preserves the 19S RP. Except for four cases caused by *de novo* heterozygous variants in *PSMB9* or *POMP*, the concomitant presence of two variants is required to cause the inflammatory phenotype, following a recessive monogenic — homozygous or compound heterozygous — or double heterozygosity inheritance (Table 1). This observation suggests that, apart from the four *PSMB9* and *POMP* outliers, which likely function as dominant-negative variants, most proteasome lesions can be easily rescued by the second wild-type allele, indicating that the majority of CANDLE/PRAAS are recessive disorders. Nevertheless, as discussed above, the current lack of consistency across variants makes it too premature to propose a model of genetic architecture for these diseases.

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## 162 Neurodevelopmental proteasomopathies

In 2017, a new form of NDD caused by loss-of-function heterozygous variants in the *PSMD12* proteasome gene was reported<sup>33</sup>. This came as a surprise since it was initially assumed that autoinflammation was the only phenotype conferred by proteasome variants. In this dominant neurodevelopmental proteasomopathy, originally referred to as Stankiewicz-Isidor syndrome, mild to severe intellectual disability with radial ray, cardiac and renal malformations represented, among other possible manifestations, an inconstant but evocative association<sup>34,35</sup>.

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Over the last five years, the concept of neurodevelopmental proteasomopathies has been strengthened by the identification of new variants in further proteasome genes in patients presenting with similar neurological manifestations. To date, 46 proteasome lesions in 78 individuals have been uncovered (Table 1). Up to this point, a total of six neurodevelopmental
175 proteasomopathies have been described, involving the four PSMB1/β6, PSMC1/Rpt2, PSMC3/Rpt5, and PSMD12/Rpn5 subunits (Table 2). In contrast to their autoinflammatory 176 counterparts, neurodevelopmental proteasomopathies are mostly monogenic dominant 177 disorders arising from *de novo* mutations in proteasome genes. Indeed, approximately 85% of 178 individuals suffering from these diseases carry heterozygous variants within the PSMD12 or 179 PSMC3 genes (Table 2). Recessive forms, reported in 8/78 patients with PSMB1, PSMC1, or 180 PSMC3 biallelic variants, represent only 10% of neurodevelopmental proteasomopathies. 181

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183 As shown in Table 2, the hallmark of these six disorders is mild to severe developmental 184 delay (100%), characterized by predominant speech delay (97%) and intellectual disability 185 (85%), very often accompanied by behavioral abnormalities (66%), mainly autism spectrum 186 disorder, and motor delay. Furthermore, nearly all patients (95%) display dysmorphic facial 187 features (Table 2), although these features are highly diverse and do not follow clinically 188 recognizable patterns. Besides these manifestations, the clinical spectrum of these disorders is 189 remarkably wide, and the phenotype associated with the variants varies significantly among 190 individuals. Notably, a significant proportion of patients exhibit multi-system malformations, 191 likely indicating the ubiquitous expression of the proteasome and the crucial role of the UPS 192 in human development. As shown in Table 2, these visceral malformations can affect the 193 brain, heart, and kidneys, as well as the reproductive and skeletal systems. Notably, visceral 194 malformations have not been reported in forms with PSMB1 or PSMC3 biallelic variants nor 195 with PSMC1 deletions (Table 2). However, these observations should be interpreted with 196 caution, given the limited number of cases reported for these forms. 197

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It seems that at least two of the six disorders can be considered as recognizable syndromes. 199

One of these is Stankiewicz-Isidor syndrome, which is consistently characterized by
200 intellectual disability, speech delay, multi-systemic malformations evocative of thumb
dysplasia or aplasia, as well as possible hearing impairment and signs of inflammation, such
201 as chilblains, urticarial skin rash, and congenital uveitis (Table 2). The second is the recessive
disorder associated with PSMC3 variants, which can be uniformly defined by developmental
202 delay, speech delay, early onset cataract, and sensorineural hearing loss (Table 2). Moreover,
203 both two syndromes highlight the relatively high frequency of hearing impairment observed
204 in neurodevelopmental proteasomopathies, in line with the role of the proteasome in inner ear
205 development ³⁶.
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209 A particularly intriguing observation is that mutations within the same PSMC3 gene can give
rise to two distinct clinical phenotypes. While one results in a recessive neurosensory
syndrome characterized by intellectual disability, ataxia, peripheral polyneuropathy, deafness,
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211 and cataract, attributed to a deep intronic mutation ³⁷, the other one presents a dominant form
of NDD featuring type I IFN production, caused by missense variants ³⁸. Remarkably,
genomic alterations impacting the PSMC1 gene exhibit a similar phenomenon with recessive
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213 missense variants causing a neurological syndrome typified by severe intellectual disability,
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215 chorea, and spastic tetraplegia³⁹, and heterozygous deletions leading to a milder form of NDD
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217 without motor delay ⁴⁰. Furthermore, while both dominant and recessive syndromes lead to
218 impaired neurodevelopment, it is noteworthy that the recessive variants seem to affect the
219 central nervous system (CNS) in a broader manner, not restricted to cognitive functions
220 (Table 2). However, it is important to acknowledge that the interpretation of these findings is
221 constrained by the limited number of patients. As such, further investigations with larger
222 patient cohorts are warranted to validate and strengthen these observations.
223

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Another compelling observation pertains to two patients harboring *PSMC3* heterozygous missense variants, both of whom have been documented to develop tumors ³⁸, specifically craniopharyngioma and neuroblastoma. This raises an important question regarding a potential predisposition to cancer in individuals affected by these diseases. Given the possibility of cancer susceptibility in these cases, it is worth mentioning that chronic inflammation, particularly type I IFN, is a well-known factor contributing to genomic instability and carcinogenesis ⁴¹. It is plausible that sustained inflammation may play a role in explaining the occurrence of tumors in some patients with *PSMC3* missense variants.

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As shown in Figure 2, the observation that only one (i.e., p.Tyr103His in *PSMB1/β6*) of the forty-three lesions identified in individuals with neurodevelopmental proteasomopathies affect the 20S CP strongly suggests that these disorders are essentially diseases of the 19S RP, an assumption which remains to be confirmed in future studies. One should also emphasize that the number of genes associated with neurodevelopmental proteasomopathies highlighted in this review is likely underestimated. Indeed, exome sequencing in large NDD cohorts recently suggested the implication of several additional strong candidates including *PSMA7/α4*, *PSMD10/p28*, or *PSMC5/Rpt6* whose *de novo* missense variants were found to be statistically enriched in patients ⁵,⁴²,⁴³. Likewise, haploinsufficiency of certain proteasome genes was suggested to contribute to the cognitive phenotype of several microdeletion syndromes, such as *PSMD10/p28* in Xq22.3, *PSMD11/Rpn6* in 17q11.2, or *PSMD14/Rpn11* in 2q24.2 deletions, respectively ⁴⁴⁻⁴⁸. While these studies did not conclusively establish the pathogenicity of these alterations in functional studies, the observation that most of these genes encode ATPases and non-ATPase subunits support the 19S RP hypothesis of these diseases, which is further reinforced by the ongoing constitution of NDD patient cohorts with variants in the *PSMC5* and *PSMD11* genes (manuscripts in preparation). Though this
249 specificity seems difficult to pinpoint, recent research by Sun et al ⁴⁹ could provide valuable
250 insights into the propensity of 19S subunits to initiate neurodevelopmental
251 proteasomopathies. This study indeed proposes that synapses have a significant enrichment of
252 "free" 19S RP that perform functions independent of the proteasome. Consequently, it is
253 plausible that neurons are more vulnerable to changes in the 19S subunits compared with
254 other cell types, as discussed later.

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256 As noted earlier, one major hurdle to our understanding of the molecular pathogenesis of
257 neurodevelopmental proteasomopathies remains their clinical heterogeneity, with a phenotype
258 spectrum ranging from mild neurodevelopmental impairment to severe neurosensory deficits.
259 Furthermore, the associated malformations seem variable and inconsistent, making it difficult
260 for now to identify a recognizable clinical pattern. Very few inherited variants reported for
261 these diseases reveal considerable intrafamilial variability in the severity of the
262 neurocognitive impairment they trigger ³⁵. For these reasons, and given the very limited
263 number of cases described so far, a genotype-phenotype correlation of these diseases remains
264 difficult to establish.

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266 Biomarkers for neurodevelopmental proteasomopathies

267 A common feature of all pathogenic proteasome gene variants is their propensity to generate
268 proteotoxic stress, because of proteasome loss of function. As discussed below, this is best
269 exemplified by the accumulation of ubiquitin-modified protein aggregates. It should,
270 however, be emphasized that virtually all tissues are equipped with control quality systems
271 capable of sensing and correcting protein homeostasis perturbations. Accordingly, this implies
272 that overactivation of either one of these processes may serve as potential biomarkers for
273 neurodevelopmental proteasomopathies as well (Fig. 3).
274

275 Protein aggregation

276 Ubiquitin-positive inclusions have been consistently documented in various cell types across
277 individuals carrying proteasome variants, regardless of their clinical phenotype 32–34,37,38,50–57.

278 These ubiquitin-modified aggregates are known to originate primarily from defective
279 ribosomal products, namely newly synthesized proteins that have failed to reach their native
280 and functional three-dimensional conformation 58. It is estimated that 20% to 30% of the
281 translation products are defective ribosomal products destined for degradation 59, thereby
282 implying that tissues with a high demand for protein synthesis are more sensitive to
283 proteasome dysfunction and proteotoxic stress. Notably, recent studies have shown that the
284 brain, liver, and pancreas are among the organs with the highest rates of protein synthesis in
285 the human body 60,61. As such, uncontrolled accumulation of ubiquitin-protein conjugates in
286 the CNS may represent one of the most reliable biomarkers of these rare diseases.
287

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288 Increased autophagy

289 Not surprisingly, dysfunction of the UPS has been shown to stimulate the second main
290 degradative pathway in eukaryotic cells, the autophagy-lysosomal pathway 62. Autophagy is a
291 highly conserved intracellular machinery that sequesters cytosolic substrates within
292 autophagosomes prior to their degradation in lysosomes 63,64. Depending on the presence of
293 recognition elements carried by substrates, autophagy may be selective or non-selective. For
294 instance, selectivity may be achieved via the modification of cargos with Lys63-linked
295 polyubiquitin chains which are then recognized by autophagy receptors such as SQSTM1/p62
296 65. The latter, in turn, delivers ubiquitinated substrates to nascent autophagosomes thanks to
297 its ability to interact with LC3 precursors and/or processed forms 66. The crosstalk between
298 UPS and autophagy is largely regarded as a compensatory mechanism enabling the cell to
cope with accumulating ubiquitin-protein conjugates when either one of these systems becomes defective. The mechanisms by which UPS impairment stimulates autophagy are diverse and rely on both gene expression and post-translational modifications. For instance, proteasome inhibition induces the expression of key components of the autophagy pathway via transcription factor EB<sup>67-70</sup>. In addition, proteasome impairment activates the autophagy-lysosomal pathway by promoting the phosphorylation of SQSTM1/p62 at Ser403 by various kinases, including PINK1, p38δ, or TBK1<sup>71-73</sup>, a process that increases the binding affinity of SQSTM1/p62 for its ubiquitin-modified cargos<sup>74</sup>. A third mechanism involves mTOR down-regulation, which occurs following proteasome inhibition due to a decreased supply of peptides and amino acids<sup>75</sup>. Reduced mTOR signaling then stimulates autophagy by shifting the phosphorylated forms of the inhibitory kinase ULK1 to the unphosphorylated ones<sup>76</sup>.

Importantly, the substrate spectrum of autophagy is by far larger than that of the UPS, as it includes intracellular organelles, such as ribosomes and mitochondria<sup>77</sup>. Accordingly, elevated degradation rates of mitochondria by autophagy, a process referred to as "mitophagy", have been frequently observed in cells treated with proteasome inhibitors<sup>78</sup>.

Supporting this notion, we could also show that individuals with Stankiewicz-Isidor syndrome exhibited a higher mitochondrial turnover than healthy individuals<sup>34</sup>. Given the importance of mitochondrial respiration in neuronal function<sup>79</sup>, it is highly likely that persistent mitophagy caused by proteasome loss of function variants might be a key driver in the pathogenesis of neurodevelopmental proteasomopathies.

## NRF1 activation

Besides alerting autophagy, defective proteasomes can augment their own synthesis, a process that relies on the endoplasmic reticulum (ER) membrane-resident protein NRF1 (also referred
to as TCF11). NRF1 is a highly glycosylated short-lived transcription factor with a half-life of approximately 12 min under normal conditions<sup>80</sup>. Upon proteasome impairment, NRF1 becomes stabilized and undergoes proteolytic cleavage by the aspartyl protease DDI2, whereby the C-terminal part translocates into the nucleus and induces the transcription of proteasome genes<sup>80</sup>. In this regard, we were able to demonstrate that the NRF1-DDI2 axis is persistently activated in patients carrying PSMC3 variants<sup>37</sup>, a process creating a vicious circle in which defective proteasomes up-regulate themselves. Interestingly, target genes of NRF1 also include genes related to autophagy (i.e., SQSTM1/p62) and mitophagy<sup>81</sup>. In addition, in the nucleus, NRF1 represses the expression of SOAT1, a gene coding for acyl-coenzyme A: cholesterol acyltransferase 1, an enzyme that catalyzes cholesterol esterification<sup>82</sup>. This observation has led to the concept that NRF1 may act as a cholesterol sensor, being retained at the ER under conditions of high membrane cholesterol concentrations, thereby reducing the levels of free cholesterol. In addition, Bartelt and colleagues have recently shown that cold temperature activates NRF1 to induce a transcriptional program promoting brown adipose tissue formation<sup>83</sup>.

Altogether, these studies point to an unexpected cross-regulation of proteasome function and lipid metabolism associating proteasome loss of function with increased mobilization of free cholesterol. This notion is in line with the fact that patients with CANDLE/PRAAS suffer from lipodystrophy<sup>32</sup>. Intriguingly, individuals suffering from neurodevelopmental proteasomopathies seem clinically devoid of lipid metabolism alterations. Their skeletal malformations, however, may be related to impaired brown adipose tissue function, whose proper functioning has been positively correlated with bone anabolism<sup>84,85</sup>.

348 Activation of the heat-shock response
Early studies have revealed that cells treated with proteasome inhibitors rapidly mount a heat-shock response<sup>86</sup>. Heat-shock response is traditionally viewed as a highly conserved process destined to induce the transcription of genes coding for stress proteins and heat-shock proteins in order to preserve protein homeostasis under challenging conditions. Prominent heat shock proteins induced by the heat-shock response include heat shock protein 70 and heat shock protein P90, whose function consists of protecting and/or refolding proteins using energy provided by ATP hydrolysis. Activation of the heat-shock response relies on the heat-shock transcription factor 1 which, under normal conditions, is retained in the cytosol. As of now, the exact molecular mechanisms responsible for heat-shock transcription factor 1 translocation into the nucleus in response to proteasome inhibition remain unknown.

Interestingly experiments in yeast have revealed that, besides molecular chaperones, target genes of heat-shock transcription factor 1 include components of the UPS<sup>87,88</sup> among which the ubiquitin E3 ligases Hul5, Rsp5, and UBE2O<sup>89-91</sup>. Of note, the mammalian ortholog of yeast Hul5, UBE3C, restores ubiquitin chains on partially proteolyzed substrates at proteasomes, a process understood to augment the processivity of proteasomes<sup>92,93</sup>. Similarly, the mammalian ortholog Rsp5, NEDD4 supports protein breakdown by shuttling ubiquitin-modified protein to proteasomes for their subsequent degradation. Finally, heat-shock transcription factor 1-induced genes include the ubiquitin fusion genes UBB, UBC, UBA52, and RPS27A<sup>94</sup>, a process likely destined to support ubiquitin conjugation. It is worth noting that, since UBA52 and RPS27A are fused with ribosomal proteins, the processing of these gene products leads to the release of RPL40 and RPS27a. The up-regulation of these proteins may also serve as an indication of proteasome loss of function.

371

372 Activation of the unfolded protein response and integrated stress response
Protein homeostasis is constantly monitored at the ER from which defective proteins are transported back to the cytosol for degradation. This process is named ER-associated degradation⁹⁵. Any decline in proteasome activity is accompanied by dysfunction of ER-associated degradation and concomitant accumulation of misfolded and/or damaged protein within the ER lumen. This agglomeration of protein aggregates in the ER activates the three ER membrane-resident receptors ATF6, PERK, and IRE1⁹⁶. These, in turn, trigger the so-called unfolded protein response, namely a compensatory transcriptional program which primarily aims at up-regulating molecular chaperones and ER-associated degradation components and whose cellular aspects have been already discussed elsewhere⁹⁷. Another key feature of the unfolded protein response is its ability to stop protein biosynthesis to prevent protein burden exacerbations. This translational arrest is mediated by PERK, which phosphorylates the initiation translation factor eIF2α. This post-translational modification inhibits GDP/GTP exchange by eIF2B⁹⁸. Importantly, eIF2α phosphorylation can also be catalyzed by three additional cytosolic serine-threonine kinases: general control non-derepressible-2, protein kinase R, and haem-regulated inhibitor. These kinases are part of the integrated stress response, a stress pathway activated in response to various pathogenic and/or proteotoxic stimuli⁹⁹. The relevance of the integrated stress response, protein kinase R in particular, as a reliable diagnostic biomarker for proteasome dysfunction has been recently highlighted in both CANDLE/PRAAS and neurodevelopmental proteasomopathies³⁸,¹⁰⁰.

## Type I IFN signatures

In this regard, protein kinase R is even more interesting, since besides phosphorylating eIF2α, it triggers a signaling cascade leading to the transcription of type I IFN genes¹⁰¹.

Accordingly, proteasome inhibition typically results in the up-regulation of type I IFN-stimulated genes such as ISG15, IFI44L, and IFIT1 in vitro in various cell types¹⁰²⁻¹⁰⁴.
Likewise, CANDLE/PRAAS and neurodevelopmental proteasomopathies have been consistently associated with persistent peripheral blood type I IFN gene signatures 32,34,38,50,52,53,55,105,106. Interestingly, Davidson et al recently highlighted the critical role of protein kinase R in initiating type I IFN response in these patients 100. In this study, the authors show that impaired protein breakdown is associated with cytosolic accumulation of IL-24, which is then sensed as a danger signal by protein kinase R. However, this notion presupposes that autoinflammation triggered by proteasome dysfunction is limited to cells expressing IL-24. Although the activation of type I IFN responses aligns with CANDLE/PRAAS phenotypes, it does not correspond to the absence of typical clinical inflammation signs observed in patients with neurodevelopmental proteasomopathies. The discordance is not a completely novel paradox, as similar observations have been made in patients with Aicardi-Goutières syndrome and Down syndrome who exhibit increased IFN signaling despite lacking inflammatory symptoms 107.

411

## 412 Molecular pathogenesis of neurodevelopmental proteasomopathies

It has long been known that proteasome dysfunction affects both development and neuronal differentiation and/or function. This has been particularly well exemplified in animal models, notably mice in which knockout of the subunit genes *Psmd4*, *Psmd11*, *Psmg1*, *Psmc3*, and *Psmc4* is embryonic lethal (Table 3) 108–111. Inevitably, conditional knockout of the *Psmg1*, *Psmc1*, *Psmc4*, *Psmd4*, and *Psmd14* genes have been associated with severe neurological phenotypes characterized by brain atrophy, malformations, neurodegeneration, or neuron loss in various mouse and/or *Drosophila* models 39,110,112–117. Supporting the importance of active proteasome function for CNS function, knockdown of the *Psmd11* and *Psmc5* genes resulted in growth retardation in mice and decreased learning ability in rats, respectively 109,116.

422
Remarkably, while depletion of the immunoproteasome subunits *Psmb8* and/or *Psmb9* is primarily associated with MHC class I antigen presentation defects (Table 3), it fails to replicate the phenotype observed in CANDLE/PRAAS patients in terms of systemic autoinflammation. However, comprehensive investigations of the CNS in these mice revealed a higher production of pro-inflammatory cytokines by astrocytes and/or microglia cells (Table 3), indicating the occurrence of neuroinflammation. Most importantly, mice with a knockout of *Psmb8* or *Psmb9* exhibit some of the manifestations observed in patients with neurodevelopmental proteasomopathies, including dysfunctional behavior<sup>118,119</sup>. These studies suggest that the overlap between autoinflammatory and neurodevelopmental proteasomopathies may be larger than initially assumed. Interestingly, as illustrated in Table 3, mice with a tissue-specific conditional knockout of the Psmc4 subunit exhibited muscle atrophy, thus linking 19S RP deficiency to reduced muscle tone.

Collectively, these mouse models underscore the significant role of proteasomes in both brain development and function. Nevertheless, our understanding of the pathogenesis of neurodevelopmental disorders remains rudimentary. Investigating the molecular mechanisms underlying these diseases is indeed hindered by the massive proteome changes occurring because of global impairment of protein breakdown. Taking this into account, the simplest hypothesis that could explain the neurological phenotype of individuals harboring proteasome loss-of-function variants is that such accumulation of protein aggregates — regardless of their origin — cannot be tolerated by the CNS. However, this would imply that the CNS is less efficient in coping with protein burden compared with other organs, an assumption for which there is little evidence thus far. Instead, it is tempting to speculate that defective proteasomes in these patients lead to the persistent expression and/or stabilization of critical regulators involved in developmental and neuronal pathways. As shown in Table 4, prominent
proteasome substrates expressed in neurons include Hes1, Mov-10, Dab1, Cdk5 activator p35, Limk1, and Rap2a, all involved to varying degrees in the regulation of neurodevelopment. Of particular interest in this group is the negative regulator Limk1, whose persistent expression has been shown to limit axon elongation <sup>120</sup>. Likewise, impaired removal of Cdk5 activator p35 by proteasomes leads to Cdk5 hyperactivation, a process that is associated with increased neurotoxicity in postmitotic neurons <sup>121</sup>.

Importantly, the breakdown of postsynaptic proteins by the UPS in response to neural activity is also a key process of both long-term potentiation and long-term depression, namely in learning and memory <sup>122</sup>. For instance, degradation of Arc/Arg3.1 by proteasomes has been shown to preserve postsynaptic cell surface expression of AMPA-type receptors and thus maintain the strength of glutamatergic synapses resulting from long-term potentiation <sup>123</sup>.

Other proteasome substrates involved in this process include synaptophysin and Akap79/150, the latter accumulating in the brains of individuals with bipolar disorders <sup>124</sup>. As shown in Table 4, the UPS also determines the molecular composition of the postsynaptic density <sup>125</sup>, a massive and complex network made-up of membrane and cytosolic scaffolding proteins supporting synaptic interactions <sup>126</sup>. Prime examples of postsynaptic density components targeted for proteasome-mediated degradation include PSD-95 and GKAP during dendritic spine remodeling. Besides, neurotransmitter receptors and/or transporters have their protein turnover regulated by the UPS (Table 4), thereby suggesting that proteasome dysfunction may profoundly alter synaptic transmission.

The formal determination of whether these proteasome substrates accumulate in the brains of patients with proteasome variants remains an area for future research. However, this question may be difficult to answer given that biological samples made available for research
investigations usually consist of whole-blood specimens. Future studies will have to overcome this problem using neurons and/or brain organoids derived from induced pluripotent stem cells.

There has been a recent postulation that 19S RP exists as free and unbound complexes in both pre- and post-synaptic compartments in rats<sup>49</sup>. Remarkably, this study proposes that 19S RP, when separated from their 20S CP, exhibit a UCHL-5-mediated de-ubiquitinating activity towards substrates modified with K63-linked ubiquitin chains. As a result, they regulate the trafficking of various synaptic proteins, including AMPA receptors, in a proteasome-independent manner<sup>49</sup>. This observation represents a potentially ground-breaking development in our understanding of the molecular pathogenesis of neurodevelopmental proteasomopathies, assuming that 19S variants do alter the de-ubiquitinating activity of free 19S RP.

As discussed earlier, proteasome dysfunction is associated with the acquisition of specific molecular signatures (Fig. 3), and it is conceivable that persistent expression of either one of these biomarkers may actively participate in disease etiology. For instance, chronic type I IFN responses have been associated with various detrimental effects on CNS function, including reduced serotonin production, impaired neurogenesis, increased demyelination, and neuronal cell death<sup>127</sup>. Given the harmful impact of type I IFN on the CNS, it is reasonable to speculate that it plays a significant role as a disease driver in neurodevelopmental proteasomopathies, similar to its involvement in monogenic early-onset autoinflammatory encephalopathy Aicardi-Goutières syndrome, which is caused by mutations in the *TREX1*, *RNASEH2A*, *RNASEH2B*, or *RNASEH2C* genes<sup>128</sup>. This assumption suggests that both Aicardi-Goutières syndrome and neurodevelopmental proteasomopathies might share the
498

same underlying pathogenic determinants, although further research is required to confirm

499

this hypothesis formally.

500

501

Diagnosis of neurodevelopmental proteasomopathies

502

With the progressive implementation of whole-genome sequencing into routine clinical

503

practice, the number of new cases of neurodevelopmental proteasomopathy is expected to

504

increase in the coming years. The identification of new variants as well as the availability of

505

larger patient cohorts will undoubtedly refine the genotype-phenotype correlation of these

506

diseases. However, the greatest challenge in establishing proper diagnosis will remain the

507

reclassification of variants of unknown significance (VUS), which typically requires

508

additional functional tests.

509

510

To date, the only way to investigate the pathogenicity of a VUS is to assess the proteasome

511

function of biological samples. Ideally, routine functional assays would allow reproducible

512

and reliable quantification of proteasome activity as well as biomarkers associated with

513

proteasome dysfunction, such as increased protein aggregation, mitophagy, and type I IFN

514

responses in blood samples. The costly and time-consuming nature of such experiments

515

revolving around methods such as native- and SDS-PAGE/Western-blotting make these

516

explorations hardly suitable for rapid diagnostic purposes and for high-throughput

517

classification of VUS.

518

519

The recent development of activity-based probes, small molecules that covalently bind to

520

active sites of enzymes may offer an interesting alternative for testing VUS from proteasome

521

genes. Using fluorescent activity-based probes, it is now indeed feasible to directly quantify

522

enzyme activity through flow cytometry <sup>129</sup>. Over the last couple of years, a large and
523 constantly increasing number of fluorescent activity-based probes have been designed to
524 monitor the activity of various proteases including proteasomes <sup>129</sup>. Although the relevance of
525 proteasome-specific activity-based probes for validating VUS remains to be fully explored,
526 they seem suitable for one-day diagnosis of neurodevelopmental proteasomopathies from
527 whole-blood samples. As discussed above, this strategy should be ideally accompanied by
528 parallel quantification of relevant biomarkers of proteasome dysfunction using the same flow
529 cytometer for a potential all-in-one platform. This objective can be achieved by using the
530 fluorescent dyes PROTEOSTAT® and MtPhagy which measure protein aggregation and
531 mitophagy respectively <sup>34,130</sup>. Besides, the use of fluorescent monoclonal antibodies directed
532 against cell surface proteins specifically up-regulated by type I IFN would allow a
533 simultaneous evaluation of type I IFN signaling activity in the same samples by flow
534 cytometry. These markers include notably SIGLEC1 (CD169), an adhesion molecule whose
535 relevance for sensing interferonopathies has already been established <sup>131</sup>. These experimental
536 approaches should provide the complementary diagnostic procedure required for testing the
537 functional significance of proteasome VUS newly identified by high throughput sequencing.
538 Future studies in this respect will aim at standardizing techniques combining both genetic and
539 functional testing for routine diagnosis of these rare diseases.

540
541 If biological samples are not readily available, another strategy involves testing whether the
542 identified VUS can restore proteasome function in established cellular models that are
543 deficient for the analyzed gene. Since all proteasome genes are essential, except for the
544 immunoproteasome ones, this approach involves designing a drug-inducible gene knockout to
545 deactivate the gene of interest at a specific point in time, shortly before the introduction of the
546 VUS. While this approach would undoubtedly clarify the pathogenicity of a given VUS, it has
547

significant limitations, including the necessity to pre-generate these models for each of the 52 proteasome genes and the possibility of overlooking tissue-specific effects.

549

550

One conceivable approach, powered by artificial intelligence, for analyzing proteasome VUS involves assessing their impact on the three-dimensional structure of the 26S proteasome.

551

552

Currently, various tools, such as SIFT, PolyPhen-2, and MutationTaster, can help predict whether a variant is likely to be pathogenic or benign based on its location and effect on the individual protein. However, a notable limitation of these tools is their inability to consider the wider implications of a particular subunit variant within the entire 26S proteasome complex. Future research will need to explore the potential of utilizing cutting-edge, deep learning-based methodologies for protein complex prediction, such as AlphaFold-Multimer<sup>132</sup>. These approaches hold indeed promise for conducting rapid *in silico* analyses, evaluating the pathogenicity of mutant subunits within the 26S proteasome complex.

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555

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558

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560

561

## Therapeutics

562

Although cognitive impairment in neurodevelopmental proteasomopathies seems predetermined at birth and incurable, there is reasonable hope for the development of therapeutic strategies controlling disease progression and/or some of the symptoms in the near future. To date, the difficulty of designing such treatments lies in the lack of relevant molecular targets, a limitation due to our poor understanding of disease pathogenesis.

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565

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567

568

Even though the contribution of persistent type IFN responses to the acquisition of neurological disability remains to be fully determined, the development of autoinflammation — even subclinical — is one manageable symptom of these diseases. Targeting inflammation is a therapeutic approach used in other genetically determined interferonopathies of the CNS,

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571
including Aicardi-Goutières syndrome, which is caused by mutations in genes coding for
proteins involved in nucleic acid processing and/or sensing<sup>133</sup>. The treatment for Aicardi-

Goutières syndrome involves the use of Janus kinase-specific inhibitors like baricitinib or
ruxolitinib to hinder sustained type I IFN signaling<sup>134-136</sup>. Although these therapies proved
effective in alleviating skin manifestations<sup>137</sup>, they were also associated with severe side
effects, including leukopenia and recurrent infections<sup>135</sup>. However, it remains unclear

whether suppressing type I IFN would lead to significant improvement in cognitive function,
and this observation raises the question of whether type I IFN is merely an epiphenomenon of
the disease. Other therapeutics for Aicardi-Goutières syndrome include reverse transcriptase
inhibitors and neutralizing antibodies<sup>133</sup>. The full extent of the latter's potential relevance and
applicability in the context of neurodevelopmental proteasomopathies is still awaiting
comprehensive exploration.

In general, future treatment strategies should aim to target upstream type I IFN responses and
seek to restore proteasome function. In this regard, one approach for restoring the activity of
proteasomes with mutated subunits is to take advantage of the physiological processes up-
regulating proteasome activity *in vivo*. However, our current knowledge about these
mechanisms originates from a handful of studies only, a major drawback limiting the number
of druggable pathways so far (Table 5).

For instance, cAMP-dependent protein kinase A and cGMP-dependent protein kinase G have
been shown to accelerate the turnover of misfolded proteins by phosphorylating specific
proteasome subunits<sup>138-140</sup>. These observations provided a new basis for targeting enzymes
regulating the intracellular pools of cAMP and cGMP second messengers in order to
maximize proteasome function. As such, treatments with phosphodiesterase inhibitors or
guanylyl cyclase stimulators have consistently been associated with increased proteasome function in various cell types, including neurons <sup>138-140</sup>. As shown in Table 5, regulation of proteasome activity also occurs at the level of p38 MAPK, whose inhibition by various small-molecule inhibitors such as PD169316, SB202190, and SB203580 results in increased proteasome activity <sup>141,142</sup>. Another regulator of proteasome function is the de-ubiquitinating enzyme USP14, whose inhibition by small-molecule inhibitor IU1 increases all three proteasome catalytic activities <sup>143</sup>. Although the effects of IU1 exerted on peptide hydrolysis by 20S proteasomes seem convincing, its ability to accelerate the degradation of aberrant proteins found in neurodegenerative diseases remains controversial <sup>144</sup>. Interestingly, some of the proteasome activators described so far include chemical compounds used for the treatment of neurological and psychiatric disorders. These are notably chlorpromazine and fluspirilene (Table 5), which primarily antagonize D2 dopamine receptors and whose mode of action on proteasomes remains to be fully explored. Gate opening of the 20S complex may also represent a promising strategy to force protein breakdown in cells with defective proteasomes.

As shown in Table 5, stimulators of 20S gate opening include the organic compounds AM-404, MK-886, and TCH-165, as well as recombinant proteins, among which ubiquitin-modified MUC1 and E6AP substrates. Besides organic compounds and recombinant proteins, a couple of natural substances have been shown to increase proteasome activity in vitro. As shown in Table 5, these include notably betulinic acid and curcumin, the latter impeding *PMSC3/Rpt5* phosphorylation by targeting the DYRK2 kinase <sup>145</sup>.

Altogether, these studies offer an array of options for restoring proteasome function and combating protein aggregation in neurodevelopmental proteasomopathies. Future studies need to address the relevance of these strategies in cells directly isolated from patients, as well as in preclinical models.
622

623

**Conclusion**

624

Neurodevelopmental proteasomopathies constitute an emerging group of Mendelian diseases, yet their complete clinical and molecular characterization remains an ongoing endeavor.

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626

Unfortunately, our understanding of their molecular pathogenesis is hampered by the implication of proteasomes in multiple — if not all — basic cellular processes. Although studies employing proteasome inhibitors have reliably described the primary cellular consequences of impaired protein breakdown, including proteotoxic stress, sterile

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inflammation, and remodeling of autophagy and lipid flux, these alone cannot account for the heterogeneity in phenotypes among neurodevelopmental proteasomopathies. Moreover, they offer limited explanations for the significant clinical variation observed within the broader category of proteasomopathies, which encompasses conditions like CANDLE/PRAAS. This leads to the hypothesis that variants in proteasome genes may exert effects extending far beyond their influence on proteasome catalytic activity. For instance, these additional effects could impact the stability of proteasomes, as well as their localization, post-translational modifications, or interactome. This point might be difficult to address considering the very low number of available biological samples worldwide and the rarity of the disease. In the meantime, research should take advantage of the increasing number of biomarkers associated with proteasome dysfunction for screening and therapeutic purposes.

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**Author contributions**

643

Conceptualization: S.C., S.B., S.K., and F.E.; data curation: S.C. W.D., V.V., and F.E.;

644

writing—original draft preparation: S.C.; writing—review and editing: S.M., B.I. S.K., and

645

F.E.; funding acquisition: S.B. and S.K. All authors read and agreed to the published version of the manuscript.

646
647

648 Acknowledgements

649 We gratefully acknowledge the support received for this work from the European Joint

650 Programme on Rare Diseases (EJP RD) under the project "UPS-NDDiag," as well as the
651 Agence Nationale de la Recherche (ANR) under the project ANR-21-CE17-0005.

652 Additionally, we extend our appreciation to Mutuelles AXA for their generous support. FE is
653 a recipient of an I-site NExT Junior Talent.

654

655 Conflict of interests

656 The authors declare no conflict of interests.

657

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1117 178. Hung AY, Sung CC, Brito IL, Sheng M. Degradation of postsynaptic scaffold GKAP
1118 and regulation of dendritic spine morphology by the TRIM3 ubiquitin ligase in rat
1119 hippocampal neurons. *PLoS One*. 2010;5(3):e9842.

1120 179. Konishi Y, Stegmüller J, Matsuda T, Bonni S, Bonni A. Cdh1-APC controls axonal
1121 growth and patterning in the mammalian brain. *Science*. 2004;303(5660):1026-1030.

1122 180. van Roessel P, Elliott DA, Robinson IM, Prokop A, Brand AH. Independent regulation
1123 of synaptic size and activity by the anaphase-promoting complex. *Cell*. 2004;119(5):707-
1124 718.

1125 181. Colledge M, Snyder EM, Crozier RA, et al. Ubiquitination regulates PSD-95 degradation
1126 and AMPA receptor surface expression. *Neuron*. 2003;40(3):595-607.

1127 182. Ito H, Morishita R, Noda M, Ishiguro T, Nishikawa M, Nagata KI. The synaptic
1128 scaffolding protein CNKSR2 interacts with CYTH2 to mediate hippocampal granule cell
1129 development. *J Biol Chem.* 2021;297(6):101427.

1130 183. Chutabhakdikul N, Surakul P. Prenatal stress increased Snk Polo-like kinase 2, SCF β-TrCP ubiquitin ligase and ubiquitination of SPAR in the hippocampus of the offspring at
1131 adulthood. *Int J Dev Neurosci.* 2013;31(7):560-567.

1133 184. Wang X, Trotman LC, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase
1134 for PTEN. *Cell*. 2007;128(1):129-139.
1135 185. Guo L, Wang Y. Glutamate stimulates glutamate receptor interacting protein 1 degradation by ubiquitin-proteasome system to regulate surface expression of GluR2. Neuroscience. 2007;145(1):100-109.

1138 186. Chin LS, Vavalle JP, Li L. Staring, a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation. J Biol Chem. 2002;277(38):35071-35079.

1140 187. de la Rocha-Muñoz A, Núñez E, Arribas-González E, López-Corcuera B, Aragón C, de Juan-Sanz J. E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2. Sci Rep. 2019;9(1):14944.

1143 188. Moriyoshi K, Iijima K, Fujii H, Ito H, Cho Y, Nakanishi S. Seven in absentia homolog 1A mediates ubiquitination and degradation of group 1 metabotropic glutamate receptors. Proc Natl Acad Sci U S A. 2004;101(23):8614-8619.

1146 189. Salinas GD, Blair LA, Needleman LA, et al. Actinfilin is a Cul3 substrate adaptor, linking GluR6 kainate receptor subunits to the ubiquitin-proteasome pathway. J Biol Chem. 2006;281(52):40164-40173.

1149 190. Kato A, Rouach N, Nicoll RA, Bredt DS. Activity-dependent NMDA receptor degradation mediated by retrotranslocation and ubiquitination. Proc Natl Acad Sci U S A. 2005;102(15):5600-5605.

1152 191. Jurd R, Thornton C, Wang J, et al. Mind bomb-2 is an E3 ligase that ubiquitinates the N-methyl-D-aspartate receptor NR2B subunit in a phosphorylation-dependent manner. J Biol Chem. 2008;283(1):301-310.

1155 192. Ma P, Wan LP, Li Y, et al. RNF220 is an E3 ubiquitin ligase for AMPA receptors to regulate synaptic transmission. Sci Adv. 2022;8(39):eabq4736.

1157 193. Fu AKY, Hung KW, Fu WY, et al. APCCdh1 mediates EphA4-dependent downregulation of AMPA receptors in homeostatic plasticity. Nat Neurosci. 2011;14(2):181-189.
1160 194. Zhu J, Lee KY, Jewett KA, Man HY, Chung HJ, Tsai NP. Epilepsy-associated gene

1161 Nedd4-2 mediates neuronal activity and seizure susceptibility through AMPA receptors.

1162 *PLoS Genet.* 2017;13(2):e1006634.

1163 195. Lin A, Hou Q, Jarzylo L, et al. Nedd4-mediated AMPA receptor ubiquitination regulates
1164 receptor turnover and trafficking. *J Neurochem.* 2011;119(1):27-39.

1165 196. Schwarz LA, Hall BJ, Patrick GN. Activity-dependent ubiquitination of GluA1 mediates
1166 a distinct AMPA receptor endocytosis and sorting pathway. *J Neurosci.*

1167 2010;30(49):16718-16729.

1168 197. Lussier MP, Nasu-Nishimura Y, Roche KW. Activity-dependent ubiquitination of the
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1170 198. Lussier MP, Herring BE, Nasu-Nishimura Y, et al. Ubiquitin ligase RNF167 regulates
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1172 2012;109(47):19426-19431.

1173 199. Saliba RS, Michels G, Jacob TC, Pangalos MN, Moss SJ. Activity-dependent
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1176 200. Teng Y, Rezvani K, De Biasi M. UBXN2A regulates nicotinic receptor degradation by
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1181 202. Rondou P, Haegeman G, Vanhoenacker P, Van Craenenbroeck K. BTB protein KLHL12
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1185 2017;1861(4):892-899.
1186

1187 204. Njomen E, Osmulski PA, Jones CL, Gaczynska M, Tepe JJ. Small molecule modulation of proteasome assembly. *Biochemistry*. 2018;57(28):4214-4224.
1188

1189 205. Jones CL, Njomen E, Sjögren B, Dexheimer TS, Tepe JJ. Small molecule enhancement of 20S proteasome activity targets intrinsically disordered proteins. *ACS Chem Biol.*
1190 2017;12(9):2240-2247.
1191

1192 206. Fiolek TJ, Keel KL, Tepe JJ. Fluspirilene analogs activate the 20S proteasome and overcome proteasome impairment by intrinsically disordered protein oligomers. *ACS Chem Neurosci.* 2021;12(8):1438-1448.
1193
1194

1195 207. Fiolek TJ, Magyar CL, Wall TJ, et al. Dihydroquinazolines enhance 20S proteasome activity and induce degradation of α-synuclein, an intrinsically disordered protein associated with neurodegeneration. *Bioorg Med Chem Lett.* 2021;36:127821.
1196
1197

1198 208. Santoro AM, Lanza V, Bellia F, et al. Pyrazolones activate the proteasome by gating mechanisms and protect neuronal cells from β-amyloid toxicity. *ChemMedChem.*
1199 2020;15(3):302-316.
1200

1201 209. Huang L, Ho P, Chen CH. Activation and inhibition of the proteasome by betulinic acid and its derivatives. *FEBS Lett.* 2007;581(25):4955-4959.
1202

1203 210. Katsiki M, Chondrogianni N, Chinou I, Rivett AJ, Gonos ES. The olive constituent oleuropein exhibits proteasome stimulatory properties *in vitro* and confers life span extension of human embryonic fibroblasts. *Rejuvenation Res.* 2007;10(2):157-172.
1204
1205

1206 211. Coleman RA, Trader DJ. Development and application of a sensitive peptide reporter to discover 20S proteasome stimulators. *ACS Comb Sci.* 2018;20(5):269-276.
1207
1208 212. Bech-Otschir D, Helfrich A, Enenkel C, et al. Polyubiquitin substrates allosterically
1209 activate their own degradation by the 26S proteasome. *Nat Struct Mol Biol.*
1210 2009;16(2):219-225.

1211 213. Peth A, Besche HC, Goldberg AL. Ubiquitinated proteins activate the proteasome by
1212 binding to Usp14/Ubp6, which causes 20S gate opening. *Mol Cell.* 2009;36(5):794-804.

1213 214. Lee D, Takayama S, Goldberg AL. ZFAND5/ZNF216 is an activator of the 26S
1214 proteasome that stimulates overall protein degradation. *Proc Natl Acad Sci U S A.*
1215 2018;115(41):E9550-E9559.

1216

1217 **Figure legends**

1218 **Figure 1** Structural organization of 26S proteasome complexes. The 26S proteasome consists
1219 of one 20S core particle (CP) associated with one end of the 19S regulatory particle (RP),
1220 which is further composed of a base and a lid, as indicated. The base of the 19S RP comprises
1221 six ATPase subunits (PSMC1 to PSMC6) and the non-ATPase subunits PSMD1, PSMD2,
1222 PSMD4, and ADRM1. The lid of the 19S RP contains the non-ATPase subunits PSMD3,
1223 PSMD6, PSMD7, PSMD8, PSMD11, PSMD12, PSMD13, PMSD14, and SEM1, as
1224 indicated. The 20S CP contains two heptameric α-rings and two heptameric β-rings, housing
1225 the standard subunits PSMA1 to PSMA7 and PSMB1 to PSMB7, respectively. In
1226 spermatoproteasomes, which are localized in the testis, PSMA7 is replaced by PSMA8. In
1227 immunoproteasomes, the catalytic standard subunits PSMB5, PSMB6, and PSMB7 are
1228 replaced by the inducible subunits PSMB8, PSMB9, and PSMB10, respectively.
1229 Thymoproteasomes in the thymus consist of immunoproteasomes in which PSMB8 is
1230 replaced by PSMB11. Eventually, proteasome activators (PA) bind to 26S proteasomes to
1231 form hybrid proteasomes. These PA include PA28-αβ, composed of PSME1 and PSME2
1232 subunits, PA28-γ, made up of PSME3 subunits, or the PA200 protein, as indicated.

1233
1234

**Figure 2** Localisation of the proteasome subunits subjected to genetic lesions in proteasomopathies. Representation of the mutant proteasome subunits found in CANDLE/PRAAS (red) and neurodevelopmental proteasomopathies (pink) within the 26S proteasome complex using the structure of Zhu et al (PDB ID: 7QY7, to be published).

1235

1236

1237

1238

1239

**Figure 3** Proteasome dysfunction results in several cellular consequences which can be used as biomarkers for screening proteasomopathies. Proteasome carrying loss-of-function mutant subunits (red) exhibit decreased activity which ultimately results in increased protein aggregation (red), as indicated. Compromised proteasome function is typically accompanied by parallel engagement of adaption programs (pink) including a rise of autophagy, mitophagy, proteasome contents, type I IFN, cholesterol esterification, ubiquitin, and molecular chaperone synthesis as well as reduction of protein biosynthesis. Both protein aggregation and compensatory pathways can be used as indicators for proteasomopathies.

1240

1241

1242

1243

1244

1245

1246

1247
1248 **Table 1** Loss-of-function variants in associated proteasome genes found in autoinflammatory

1249 (CANDLE/PRAAS) and neurodevelopmental proteasomopathies.

<table><thead><tr><th></th><th>Gene</th><th>Function</th><th>Gene variant</th><th>Protein variant</th><th>Cases</th><th>Inheritance</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="15">Autoinflammatory proteasomopathies</td><td rowspan="15">Monogenic</td><td rowspan="15">PSMB4</td><td>c.-9G&gt;A</td><td>5' UTR region</td><td rowspan="4">1</td><td rowspan="15">Compound heterozygosity</td><td>32</td></tr><tr><td>c.634_642del</td><td>p.(D212_V214del)</td><td>50</td></tr><tr><td>c.231del</td><td>p.(L78Wfs*31)</td><td>50</td></tr><tr><td>c.494+17A&gt;G</td><td>p.(?)</td><td>50</td></tr><tr><td>c.590G&gt;T</td><td>p.G197V</td><td>51</td></tr><tr><td>c.602G&gt;T</td><td>p.G201V</td><td>52</td></tr><tr><td>c.405C&gt;A</td><td>p.C135*</td><td>146</td></tr><tr><td>c.224C&gt;T</td><td>p.T75M</td><td>30</td></tr><tr><td>c.349A&gt;G</td><td>p.M117V</td><td>147</td></tr><tr><td>c.271G&gt;A</td><td>p.A92T</td><td>148</td></tr><tr><td>Undisclosed</td><td>Undisclosed</td><td>149</td></tr><tr><td rowspan="10">PSMB8</td><td rowspan="10">β-subunit (catalytic)</td><td>c.224C&gt;T</td><td>p.T75M</td><td rowspan="4">1</td><td rowspan="10">Compound heterozygosity</td><td>150</td></tr><tr><td>c.271G&gt;A</td><td>p.A92T</td><td>151</td></tr><tr><td>c.373C&gt;T</td><td>p.R125C</td><td>152</td></tr><tr><td>c.355G&gt;A</td><td>p.D119N</td><td>153</td></tr><tr><td>c.163C&gt;T</td><td>p.Q55*</td><td rowspan="4">1</td><td rowspan="4">Compound heterozygosity</td><td>153</td></tr><tr><td>c.352T&gt;C</td><td>p.S118P</td><td>154</td></tr><tr><td>c.275C&gt;T</td><td>p.A92V</td><td>154</td></tr><tr><td>c.313A&gt;C</td><td>p.K105Q</td><td>154</td></tr><tr><td>c.602G&gt;T</td><td>p.Gly201Val</td><td>154</td></tr><tr><td>c.389delT</td><td>p.129Argfs*27</td><td>154</td></tr></tbody></table>
<table><tbody><tr><td>PSMB9</td><td>β-subunit<br>(catalytic)</td><td>c.467G>A</td><td>p.G156D</td><td>3</td><td>Heterozygosity</td><td>105<br>53</td></tr><tr><td>PSMB10</td><td>β-subunit<br>(catalytic)</td><td>c.41T>C</td><td>p.F14S</td><td>1</td><td>Homozygosity</td><td>54</td></tr><tr><td></td><td></td><td>c.344_345ins<br>TTTGA</td><td>p.E115Dfs*20</td><td>1</td><td></td><td></td></tr><tr><td>POMP</td><td>Proteasome<br>assembly<br>factor</td><td>c.342_348del<br>nsACC</td><td>p.F114Lfs*18</td><td>1</td><td>Heterozygosity</td><td>55</td></tr><tr><td></td><td></td><td>c.334_335del<br>AT</td><td>p.I112Wfs*3</td><td>1</td><td></td><td></td></tr><tr><td></td><td></td><td>c.326dupA</td><td>p.D109Efs*2</td><td>1</td><td></td><td>155</td></tr><tr><td>PSMG2</td><td>Proteasome<br>assembly<br>factor</td><td>c.666_667del<br>GT<br>c.675T>G</td><td>p.Y223Sfs*2<br>p.N225K</td><td>1</td><td>Compound<br>heterozygosity</td><td>56</td></tr><tr><td>Double heterozygosity</td><td>β-subunit</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PSMB4</td><td>(non-catalytic)</td><td>c.666C>A</td><td>p.Y222*p.K105Q</td><td>2</td><td></td><td></td></tr><tr><td>PSMB8</td><td>β-subunit<br>(catalytic)</td><td>c.313A>C</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td>c.224C>T</td><td>p.T75M</td><td>1</td><td>Double<br>heterozygosity</td><td>32</td></tr><tr><td>PSMB8</td><td>β-subunit</td><td>c.404+2T>C</td><td>p.H111Ffs*10</td><td></td><td></td><td></td></tr><tr><td>PSMA3</td><td>β-subunit</td><td>c.224C>T<br>c.696_698del</td><td>p.T7E5M<br>p.R233del</td><td>1</td><td></td><td></td></tr><tr><td>PSMB9</td><td>β-subunit</td><td>c.494G>A</td><td>p.G165D</td><td>2</td><td></td><td></td></tr><tr><td>PSMB4</td><td>β-subunit</td><td>c.44insG</td><td>p.P16Sfs*45</td><td></td><td></td><td></td></tr><tr><td>Neuro<br>devel<br>Mono</td><td>β-subunit</td><td>c.307T>C</td><td>p.Y103H</td><td>2</td><td>Homozygosity</td><td>156</td></tr></tbody></table>
<table><tbody><tr><td rowspan="10">PSMC1</td><td rowspan="10">ATPase subunit</td><td>c.983T>C</td><td>p.I328T</td><td>1</td><td>Homozygosity</td><td>39</td></tr><tr><td>Deletion</td><td>Expected haploinsufficiency</td><td>3</td><td>Heterozygosity</td><td>40</td></tr><tr><td>c.1127+337A</td><td>p.S376Rfs15*</td><td>3</td><td>Homozygosity</td><td>37</td></tr><tr><td>&gt;G</td><td></td><td></td><td></td><td></td></tr><tr><td>c.511C>T</td><td>R171W</td><td>1</td><td></td><td></td></tr><tr><td>c.523A&gt;G</td><td>M175V</td><td>1</td><td></td><td></td></tr><tr><td>c.686C&gt;T</td><td>P229L</td><td>1</td><td></td><td></td></tr><tr><td>c.710C&gt;T</td><td>A237V</td><td>1</td><td></td><td></td></tr><tr><td>c.775A&gt;G</td><td>M259V</td><td>1</td><td></td><td></td></tr><tr><td>c.776T&gt;C</td><td>M259T</td><td>1</td><td></td><td></td></tr><tr><td rowspan="12">PSMC3</td><td rowspan="12">ATPase subunit</td><td>c.782T&gt;C</td><td>I261T</td><td>6</td><td></td><td></td></tr><tr><td>c.784G&gt;A</td><td>G262R</td><td>1</td><td>Heterozygosity</td><td>38</td></tr><tr><td>c.806G&gt;C</td><td>R269P</td><td>1</td><td></td><td></td></tr><tr><td>c.859G&gt;C</td><td>E287Q</td><td>1</td><td></td><td></td></tr><tr><td>c.910C&gt;T</td><td>R304W</td><td>4</td><td></td><td></td></tr><tr><td>c.910C&gt;G</td><td>R304G</td><td>1</td><td></td><td></td></tr><tr><td>c.915G&gt;T</td><td>E305D</td><td>1</td><td></td><td></td></tr><tr><td>c.929T&gt;C</td><td>M310T</td><td>1</td><td></td><td></td></tr><tr><td>c.1147G&gt;A</td><td>E383K</td><td>1</td><td></td><td></td></tr><tr><td>c.367C&gt;T</td><td>p.R123*</td><td>1</td><td></td><td>35</td></tr><tr><td rowspan="3">PSMD12</td><td rowspan="3">non-ATPase subunit</td><td>c.1274T&gt;G</td><td>p.L425*</td><td>1</td><td>Heterozygosity</td><td>33</td></tr><tr><td>c.601C&gt;T</td><td>p.R201*</td><td>2</td><td></td><td>33</td></tr></tbody></table>
<table><tr><td>c.909-2A>G</td><td>p.(?)</td><td>1</td><td>33</td></tr><tr><td>Deletion</td><td>Expected haploinsufficiency</td><td>10</td><td>33<br>34</td></tr><tr><td>c.544C>T</td><td>p.R182*</td><td>1</td><td></td></tr><tr><td>c.1071_1072delAG</td><td>p.R357fs*3</td><td>1</td><td></td></tr><tr><td>c.435_438del</td><td>p.T146Kfs*3</td><td>1</td><td></td></tr><tr><td>c.937G>T</td><td>p.E313*</td><td>1</td><td></td></tr><tr><td>c.508_509del</td><td>p.Q170Gfs*40</td><td>2</td><td></td></tr><tr><td>A>T</td><td>p.L149*</td><td>1</td><td></td></tr><tr><td>c.316C>T</td><td>p.Q106*</td><td>1</td><td></td></tr><tr><td>c.1033G>T</td><td>p.E345*</td><td>1</td><td></td></tr><tr><td>c.1083+1G>A</td><td>p.(?)</td><td>1</td><td></td></tr><tr><td>c.1246C>T</td><td>p.Q416*</td><td>1</td><td>34</td></tr><tr><td>c.526del</td><td>p.S176Qfs*15</td><td>1</td><td></td></tr><tr><td>c.1162-1G>A</td><td>p.(?)</td><td>1</td><td></td></tr><tr><td>c.1300del</td><td>p.S434Hfs*2</td><td>1</td><td></td></tr><tr><td>c.795+1G>A</td><td>p.(?)</td><td>1</td><td></td></tr><tr><td>c.148_149del</td><td>p.L50Gfs*26</td><td>1</td><td></td></tr><tr><td>c.435_438del</td><td>p.T146Kfs*3</td><td>1</td><td></td></tr><tr><td>c.544C>T</td><td>p.R182*</td><td>1</td><td></td></tr><tr><td>c.1060_1061del</td><td>p.L354Efs*6</td><td>1</td><td></td></tr><tr><td>c.906C>A</td><td>p.Y302*</td><td>1</td><td></td></tr></table>
c.865C>T

p.R289*

2

57
<table><caption>1251 Table 2 Clinical features of neurodevelopmental proteasomopathies.</caption><thead><tr><th></th><th>Stankiewicz-Isidor syndrome with type I IFN production</th><th>Disease with ID, microcephaly, and developmental delay</th><th>Neurosensory syndrome combining deafness and cataract</th><th>Neurodevelopmental disorder with type I IFN production</th><th>Neurological syndrome</th><th>Developmental and language delay</th><th>Total</th></tr></thead><tbody><tr><td>Gene involved</td><td>PSMD12<br>(33/45) De novo;</td><td>PSMB1</td><td>PSMC3</td><td></td><td>PSMC1</td><td></td><td></td></tr><tr><td>Inheritance</td><td>(9/45) dominant;<br>(3/45) n.a</td><td>(2/2) Recessive</td><td>(3/3) Recessive</td><td>(21/22) De novo<br>(1/22) n.a</td><td>(3/3) Recessive</td><td>(1/3) De novo; (1/3)<br>dominant; (1/3) n.a</td><td></td></tr><tr><td>References</td><td>33–35, 57</td><td>156</td><td>37</td><td>38</td><td>39</td><td>40</td><td></td></tr><tr><td>Number of published patients</td><td>45</td><td>2</td><td>3</td><td>22</td><td>3</td><td>3</td><td>78</td></tr><tr><td>Gender (Age at last assessment, years)</td><td>24 M / 21 F (1 to 42)</td><td>2 F (22 to 35)</td><td>3 M (7 to 16)</td><td>13 M / 9 F (age n.a)</td><td>3 M (3 to 20)</td><td>3 M (3 to 6)</td><td>45 M/78<br>(57.6%)</td></tr></tbody></table>
<table><thead><tr><th>Variants</th><th>Heterozygous truncating variant</th><th>Homozygous missense</th><th>Homozygous splice variant</th><th>Heterozygous missense</th><th>Homozygous missense</th><th>Heterozygous deletions</th><th></th></tr></thead><tbody><tr><td colspan="8"><em>Neurodevelopment</em></td></tr><tr><td>Developmental delay</td><td>100% (45/45)</td><td>100% (2/2)</td><td>100% (3/3)</td><td>100% (21/21)</td><td>100% (3/3)</td><td>100% (3/3)</td><td>77/77<br>(100%)</td></tr><tr><td>Speech delay</td><td>94% (32/34)</td><td>100% (2/2)</td><td>100% (3/3)</td><td>100% (18/18)</td><td>100% (3/3)</td><td>100% (3/3)</td><td>61/63<br>(97%)</td></tr><tr><td>Motor delay</td><td>50% (17/34)</td><td>100% (2/2)</td><td>n.r</td><td>78% (14/18)</td><td>100% (3/3)</td><td>0% (0/3)</td><td>36/60<br>(60%)</td></tr><tr><td>Intellectual disability; severity</td><td>85% (34/40); mild to severe</td><td>100% (2/2); severe</td><td>100% (3/3); mild to severe</td><td>88% (15/17); n.a</td><td>100% (3/3); severe</td><td>33% (1/3); mild</td><td>58/68<br>(85%)</td></tr><tr><td>Abnormal behaviors</td><td>67% (25/37)</td><td>100% (2/2)</td><td>66% (2/3)</td><td>53% (9/17)</td><td>n.a</td><td>100% (3/3)</td><td>41/62<br>(66%)</td></tr><tr><td>Autistic features</td><td>31% (13/42)</td><td>0% (0/2)</td><td>66% (2/3)</td><td>n.a</td><td>n.a</td><td>33% (1/3)</td><td>16/50<br>(32%)</td></tr></tbody></table>
ADHD features 12% (5/42) 50% (1/2) 0% (0/3) n.a n.a 33% (1/3) 7/50 (14%)

Neurological features

Epilepsy 13% (6/45) 0% (0/2) 0% (0/3) 25% (5/20) 0% (0/3) 0% (0/3) 11/76 (14%)

Regression n.r n.r n.r n.r n.r n.r

Ataxia 100% (3/3); Spastic tetraplegia

Other peripheral 100% (3/3); chorea

neurological n.r Hypotonia 100% (2/2) polyneuropathy of n.r 100% (3/3); central n.r

findings lower limbs 66% hypotonia 100% (2/3)

21% (4/19);

Abnormal brain pineal cyst, cerebral n.a 0% (0/3) 79% (11/14); additional 100% (2/2);

MRI atrophy, description n.a ventriulomegaly

hypomyelination,
periventricular

nodular heterotopia

## Morphological features

### Intrauterine

growth 37% (9/24) n.r n.r n.r n.r n.r

restriction

Other antenatal features n.r n.r n.r n.r n.r n.r

Growth failure 40% (4/10) 100% (2/2) n.r 50% (9/18) n.r n.r 15/30 (50%)

Abnormal facial shape 97% (44/45) 100% (2/2) 100% (3/3) 89% (17/19) 100% (3/3) 66% (2/3) 71/75 (95%)

Microcephaly 21% (9/42) 100% (2/2) n.r 38% (6/16) 100% (3/3) n.r 20/63 (32%)

<figure><img src="image_4.png" /></figure>
<table><tbody><tr><td>Macrocephaly</td><td>7% (3/42)<br>61% (21/34);</td><td>0% (0/2)</td><td>n.r</td><td>13% (2/16)</td><td>0% (0/3)</td><td>n.r</td><td>5/63<br>(8%)</td></tr><tr><td>Skeletal abnormalities</td><td>including thumb anomalies: 29%<br>(10/34)</td><td>n.r</td><td>n.r</td><td>71% (10/14)</td><td>n.r</td><td>n.r</td><td></td></tr><tr><td colspan="8"><em>Sensorial features</em></td></tr><tr><td>Hearing impairment</td><td>18% (6/34)<br>74% (20/27);</td><td>100% (2/2)</td><td>100% (3/3)</td><td>8/18 (44%)</td><td>100% (3/3)</td><td>n.r</td><td>22/60<br>(37%)</td></tr><tr><td>Ophthalmological abnormalities</td><td>including strabismus, coloboma, Peters anomaly, corneal opacity</td><td>n.r</td><td>Early onset cataract: 100% (3/3);<br>strabismus: 66% (2/3)</td><td>n.a</td><td>n.r</td><td>n.r</td><td></td></tr></tbody></table>

Visceral features
<table><tbody><tr><td>Cardiac</td><td>44% (15/34)</td><td>n.r</td><td>n.r</td><td>59% (10/17)</td><td>33% (1/3)</td><td>n.r</td><td>26/54<br>(48%)</td></tr><tr><td>Renal</td><td>50% (15/30)</td><td>n.r</td><td>n.r</td><td>29% (4/14)</td><td>33% (1/3)</td><td>n.r</td><td>20/47<br>(42%)</td></tr><tr><td>Genital</td><td>40% (12/30)</td><td>n.r</td><td>n.r</td><td>n.r</td><td>100% (3/3)</td><td>n.r</td><td>15/33<br>(45%)</td></tr><tr><td>Other findings</td><td>Congenital<br>pancytopenia: 2%<br>(1/45); cleft palate:<br>2% (1/45)</td><td>n.r</td><td>Semicircular canal<br>malformations 33%<br>(1/3); depigmented<br>hairs of lower limbs<br>66% (2/3)</td><td>Tumors: 11% (2/18)<br>craniopharyngioma and<br>neuroblastoma; orofacial<br>clefts: 10% (2/19)</td><td>n.r</td><td>Sleep irregularities:<br>66% (2/3)</td><td></td></tr><tr><td colspan="9"><em>Inflammatory symptoms</em></td></tr><tr><td>Chilblains</td><td>5% (2/36)</td><td>n.r</td><td>n.r</td><td>n.r</td><td>n.r</td><td>n.r</td><td></td></tr><tr><td>Urticarial skin<br>rashes</td><td>3% (1/36)</td><td>n.r</td><td>n.r</td><td>n.r</td><td>n.r</td><td>n.r</td><td></td></tr></tbody></table>
<table><tr><td>Congenital uveitis</td><td>3% (1/36)</td><td>n.r</td><td>n.r</td><td>n.r</td><td>n.r</td><td>n.r</td></tr><tr><td>Subcutaneous calcifications</td><td>n.r</td><td>n.r</td><td>100% (3/3)</td><td>n.r</td><td>n.r</td><td>n.r</td></tr></table>

1252 Notes: n.r: not reported; n.a: not assessed.

1253
<table><caption>1254 Table 3 Mouse models for proteasome subunits.</caption><thead><tr><th>Gene</th><th>Technique</th><th>Distribution</th><th>Phenotype</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">Psmc1</td><td>Tissue-specific conditional gene knockout (−/−)</td><td>Neurons</td><td>Neurodegeneration</td><td>117</td></tr><tr><td>Conventional gene knockout (+/−)</td><td>Ubiquitous</td><td>Cell cycle defects</td><td>157</td></tr><tr><td rowspan="2">Psmc3</td><td>Conventional gene knockout (−/−)</td><td>Ubiquitous</td><td>Embryonic lethal</td><td>111</td></tr><tr><td>Conventional gene knockout (−/−)</td><td>Ubiquitous</td><td>Embryonic lethal</td><td>111</td></tr><tr><td rowspan="2">Psmc4</td><td>Tissue-specific conditional gene knockout (−/−)</td><td>Muscles</td><td>Myofiber degeneration</td><td>158</td></tr><tr><td>Tissue-specific conditional gene knockout (−/−)</td><td>Muscles</td><td>Muscle atrophy</td><td>159</td></tr><tr><td rowspan="3">Psmc6</td><td>Conventional gene knockout (−/−)</td><td>Ubiquitous</td><td>Protection against<br>ovariectomy-induced<br>osteoporosis</td><td>160</td></tr><tr><td>Conventional gene knockout (−/−)</td><td>Ubiquitous</td><td>Embryonic lethal</td><td></td></tr><tr><td>Conditional PSMD4 ubiquitin-interacting motifs<br>(UIM) knockout (−/−)</td><td>Ubiquitous</td><td>Embryonic lethal</td><td>108</td></tr><tr><td rowspan="2">Psmd4</td><td>Tissue-specific conditional PSMD4 ubiquitin-<br>interacting motifs (UIM) knockout (−/−)</td><td>Liver</td><td>Disruption of protein<br>homeostasis</td><td></td></tr><tr><td>Conventional gene knock-in</td><td>Ubiquitous</td><td>Neurodegeneration</td><td>161</td></tr><tr><td rowspan="4">Psmd11</td><td>Conventional gene knockout (−/−)</td><td>Ubiquitous</td><td>Embryonic lethal</td><td>109</td></tr><tr><td>Conventional gene knockout (+/−)</td><td>Ubiquitous</td><td>Growth retardation</td><td></td></tr><tr><td></td><td></td><td>Lipodystrophy</td><td>162</td></tr><tr><td></td><td></td><td>Bipolar cell response<br>defects</td><td>163</td></tr><tr><td rowspan="3">Psmb8</td><td>Conventional gene knockout (−/−)</td><td>Ubiquitous</td><td>Abnormal behavior</td><td>118</td></tr><tr><td></td><td></td><td>MHC class I antigen<br>presentation defects</td><td>164, 165</td></tr></tbody></table>
<table><tr><td></td><td>Conventional gene knockout (−/−)</td><td></td><td>Neuroinflammation</td><td>166</td></tr><tr><td></td><td></td><td></td><td>Neuroinflammation and neurobehavioral dysfunctions</td><td>119</td></tr><tr><td></td><td></td><td></td><td>MHC class I antigen presentation defects</td><td>164, 165</td></tr><tr><td rowspan="2">Psmb9</td><td>Conventional knock-in of a Psmb9 variant (G156D/G156D)</td><td rowspan="2">Ubiquitous</td><td>Lethal</td><td>53</td></tr><tr><td>Conventional knock-in of a Psmb9 variant (+/G156D)</td><td>Immunodeficiency</td><td>53</td></tr><tr><td rowspan="2">Psmb11</td><td>Conventional gene knockout (−/−)</td><td>Thymus</td><td>MHC class I antigen presentation defects</td><td>164, 165</td></tr><tr><td></td><td>Ubiquitous</td><td>Embryonic lethal Growth retardation and</td><td></td></tr><tr><td rowspan="2">Psmg1</td><td rowspan="2">Tissue-specific conditional gene knockout (−/−)</td><td>Brain</td><td>abnormal limb-clasping reflexes</td><td>110</td></tr><tr><td>Liver</td><td>Senescence</td><td></td></tr></table>
<table><caption>**Table 4** List of proteasome substrates involved in neurogenesis and/or neuronal function.</caption><thead><tr><th>Function</th><th>Proteasome substrate</th><th>Ubiquitin ligase(s)</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="7">Neurodevelopment</td><td>Hes1</td><td>SCF<sup>FBXL14</sup></td><td>167</td></tr><tr><td>Mov-10</td><td>CRL4<sup>DCAF12</sup></td><td>168</td></tr><tr><td>Dab1</td><td>Cul5</td><td>169</td></tr><tr><td>Cdk5 activator p35</td><td>Unknown</td><td>170, 171</td></tr><tr><td>LIM domain kinase 1</td><td>Rnf6</td><td>172</td></tr><tr><td>Rap2A</td><td>Nedd4</td><td>173</td></tr><tr><td>Arc/Arg3.1</td><td>Ube3A, Triad3A</td><td>174, 175</td></tr><tr><td rowspan="5">Synaptic plasticity</td><td>Synaptophysin</td><td>Siah1</td><td>176</td></tr><tr><td>Akap79/150</td><td>Unknown</td><td>177</td></tr><tr><td>GKAP</td><td>Trim3</td><td>178</td></tr><tr><td>Liprin-α</td><td>APC/C</td><td>179, 180</td></tr><tr><td>PSD-95</td><td>Mdm2</td><td>181</td></tr><tr><td rowspan="7">Postsynaptic scaffolding</td><td>Shank1</td><td>Unknown</td><td>177</td></tr><tr><td>CNKSR2</td><td>Smurf2</td><td>182</td></tr><tr><td>SPAR</td><td>SCF<sup>β-TRCP</sup></td><td>183</td></tr><tr><td>PTEN</td><td>Nedd4</td><td>184</td></tr><tr><td>GRIP1</td><td>Unknown</td><td>185</td></tr><tr><td>Syntaxin1</td><td>Rnf40</td><td>186</td></tr><tr><td>GlyT2</td><td>LNX1 and LNX2</td><td>187</td></tr><tr><td rowspan="4">Synaptic transmission</td><td>mGluR1 and mGluR5</td><td>Siah1A</td><td>188</td></tr><tr><td>GluK2</td><td>Cul3 actinfilin</td><td>189</td></tr><tr><td>GluN1</td><td>Fbx2</td><td>190</td></tr><tr><td>GluN2A</td><td>Mib2</td><td>191</td></tr></tbody></table>
<table><tr><td>GluA1</td><td>Nedd4, Nedd4L, APC<sup>Cdh1</sup></td><td>192–196</td></tr><tr><td></td><td></td><td>192, 197,</td></tr><tr><td>GluA2</td><td>RNF167, RNF220</td><td>198</td></tr><tr><td>GABA<sub>A</sub>Rβ3</td><td>Unknown</td><td>199</td></tr><tr><td>nAChRα3</td><td>CHIP</td><td>200</td></tr><tr><td>GlyR</td><td>Unknown</td><td>201</td></tr><tr><td>DRD4</td><td>KLHL12</td><td>202</td></tr><tr><td>DRD1, DRD5, and DRD2L</td><td>Unknown</td><td>202</td></tr></table>
<table><caption>**Table 5** List of molecules described as proteasome stimulators.</caption><thead><tr><th></th><th>Molecule</th><th>Mode of action</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="14">Chemical compounds</td><td>IU1</td><td>USP14 inhibition</td><td>143</td></tr><tr><td>Rolipram</td><td>Phosphodiesterase-4 inhibition</td><td>138</td></tr><tr><td>Cilostazol</td><td>Phosphodiesterase-3 inhibition</td><td>139</td></tr><tr><td>Sildenafil</td><td>Phosphodiesterase-5 inhibition</td><td></td></tr><tr><td>Tadalafil</td><td></td><td>140</td></tr><tr><td>BAY41-2272</td><td>Guanylyl cyclase stimulation</td><td></td></tr><tr><td>Cinaciguat</td><td></td><td></td></tr><tr><td>PD169316</td><td></td><td></td></tr><tr><td>SB202190</td><td>p38 MAPK inhibition</td><td>141</td></tr><tr><td>SB203580</td><td></td><td></td></tr><tr><td>PD169316</td><td></td><td>142</td></tr><tr><td>AM-404</td><td></td><td></td></tr><tr><td>MK-886</td><td>20S Gate opening stimulation</td><td>203</td></tr><tr><td>TCH-165</td><td></td><td>204</td></tr><tr><td>Chlorpromazine</td><td>D2 dopamine receptor blockade</td><td>205</td></tr><tr><td>Fluspirilene</td><td></td><td>206</td></tr><tr><td>Dihydroquinazolines</td><td>Trypanothione reductase inhibition</td><td>207</td></tr><tr><td rowspan="2">Natural substances</td><td>Nifenazone</td><td>Nonsteroidal anti-inflammatory drug</td><td>208</td></tr><tr><td>Betulinic acid</td><td>Unknown</td><td>209</td></tr><tr><td></td><td>Curcumin</td><td>DYRK2 inhibition</td><td>145</td></tr><tr><td></td><td>Oleuropein</td><td>Unknown</td><td>210</td></tr><tr><td></td><td>Ursolic acid</td><td>p38 NF-κB inhibition</td><td>211</td></tr></tbody></table>
<table><tr><td></td><td>Ubiquitinated</td><td></td></tr><tr><td rowspan="3">Recombinant proteins</td><td>MUC1</td><td>212</td></tr><tr><td>Ubiquitin aldehyde</td><td>20S Gate opening stimulation</td><td>213</td></tr><tr><td>Ubiquitinated E6AP</td><td></td><td>213</td></tr><tr><td></td><td>ZFAND</td><td>214</td></tr></table>
<figure><img src="image_5.png" /></figure>

Autoinflammatory proteasomopathies

Neurodevelopmental proteasomopathies
<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>