# The PTCHD1 protein: A prominent actor in brain function and in neurodevelopmental disorders

Dévina C Ung, Stéphane Martin, Yann Hérault, Frédéric Laumonnier

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Dévina C Ung, Stéphane Martin, Yann Hérault, Frédéric Laumonnier. The PTCHD1 protein: A prominent actor in brain function and in neurodevelopmental disorders. Neuroscience and Biobehavioral Reviews, 2025, 176, pp.106307. 10.1016/j.neubiorev.2025.106307. inserm-05209366

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# The PTCHD1 protein: A prominent actor in brain function and in neurodevelopmental disorders

Dévina C. Ung<sup>a,*</sup> ◆, Stéphane Martin<sup>b</sup>, Yann Hérault<sup>c,d</sup>, Frédéric Laumonnier<sup>a,e</sup>

## ARTICLE INFO

### Keywords:

PTCHD1
Neurodevelopmental disorders
Intellectual disability
Autism spectrum disorders
Synaptic proteome
Neuronal signaling pathway
Mouse models

## ABSTRACT

Neurodevelopmental disorders (NDDs) encompass a broad spectrum of conditions, including intellectual disability (ID), Attention Deficit Hyperactivity Disorders (ADHD) and autism spectrum disorders (ASD), often linked to disrupted synaptic functionality. Among the major NDD genes, *PTCHD1* (*Patched Domain Containing 1*) encodes a transmembrane protein which acts as a crucial regulator of synaptic function. Mutations and microdeletions in *PTCHD1* have been associated with global developmental delays such as ID, ASD, and behavioral abnormalities. This review explores the neurodevelopmental functions of PTCHD1, particularly its position within the synaptic environment, from the association with the postsynaptic proteome to the regulation of several major neuronal signaling pathways, cholesterol homeostasis and receptor trafficking. In a pathophysiological context, studies in *Ptchd1* knockout mouse models revealed abnormal behavioral phenotypes, linked to synaptic impairments, including reduced excitatory postsynaptic currents and altered dendritic morphology similarly to recent results on human-derived neuronal models. By synthesizing findings from genetic, molecular, and behavioral studies, this review underscores the multifaceted roles of PTCHD1 in neurodevelopment and synaptic regulation. Understanding its function in synaptic pathways may provide crucial insights into the pathophysiology of NDDs, emphasizing the need for further research to clarify its role as a potential synaptic, orphan receptor.

## 1. Introduction

Neurodevelopmental disorders (NDDs) represent a diverse spectrum of pathological conditions characterized by deficits in higher-order functions such as cognition, communication, social behavior, and motor skills. Notable among these disorders are Intellectual Disabilities (ID), Attention Deficit Hyperactivity Disorders (ADHD), and Autism Spectrum Disorders (ASD), which exhibit a significant prevalence of 10.5–13.2 % in the general population (Zablotsky et al., 2019). The etiologies of NDDs are diverse, ranging from metabolic and environmental causes to genetic origins, which account for nearly half of ID cases (Vissers et al., 2016) and 25 % of ASD cases (Gaugler et al., 2014). Recent advances in genetic screening technologies have propelled efforts to elucidate the etiologies of NDDs, resulting in the identification of

at least 1500 candidate genes associated with ID and ASD (Sys-NDD database, Kochinke et al., 2016) and highlighting substantial heterogeneity. The spectrum of clinical and genetic phenotypes associated with NDDs characterizes a multitude of rare developmental disorders that necessitate the development of personalized medicine. Nevertheless, functional investigations into these genes have unveiled a notable convergence towards common molecular pathways, including transcriptional regulation, chromatin remodeling, protein synthesis, cellular metabolism, synaptic development and function that could impact either brain early development and/or adult functioning (Krishnan et al., 2016; Satterstrom et al., 2020; Parenti et al., 2020).

Among the identified NDD genes, *PTCHD1* (*Patched Domain Containing 1*), located on the X chromosome (locus Xp22.11) emerged from the description of truncating mutations, deletions including upstream

<footnote><sup>a</sup> Université de Tours, INSERM, Imaging Brain & Neuropsychiatry iBraiN U1253, Tours 37032, France</footnote>

<footnote><sup>b</sup> Université Côte d'Azur, Inserm, CNRS, IPMC, Valbonne 06560, France</footnote>

<footnote><sup>c</sup> CNRS, INSERM, CELPHEDIA, PHENOMIN, Institut Clinique de la Souris (ICS), Université de Strasbourg, Illkirch 67404, France</footnote>

<footnote><sup>d</sup> Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS-UMR7104, INSERM U1258, Université de Strasbourg, Illkirch 67404, France</footnote>

<footnote><sup>e</sup> Service de Génétique, Centre Hospitalier Régional Universitaire, Tours 37044, France</footnote>

<footnote>* Correspondence to: UMR1253 iBraiN, Faculty of Medicine, 10 Bd Tonnellé, Tours Cedex 37032, France.
*E-mail addresses:* devina.legrand-ung@inserm.fr (D.C. Ung), martin@ipmc.cnrs.fr (S. Martin), herault@igbmc.fr (Y. Hérault), frederic.laumonnier@univ-tours.fr (F. Laumonnier).</footnote>
and promoter regions of *PTCHD1* in about 1 % of male patients with ASD and ID (Marshall et al., 2008; Noor et al., 2010; Pinto et al., 2010; Chaudhry et al., 2015; Wells et al., 2016; Pastore et al., 2025). Phenotype-genotype studies concluded that *PTCHD1* gene variations result in an X-linked non-syndromic NDD characterized by global developmental delay, variable features of ID and/or ASD, hypotonia, motor incoordination, and predominantly dysmorphic features (Filges et al., 2011; Pastore et al., 2022). Additionally, patients may present behavioral and/or psychiatric issues, including attention deficit with or without hyperactivity (ADHD), sleep disruptions as well as aggressive and/or impulsive behaviors (Chaudhry et al., 2015; Wells et al., 2016; Pastore et al., 2022).

In mammals, the *PTCHD1* gene encodes a protein with a predicted secondary structure composed of 12 transmembrane domains (TMD) similarly to the PATCHED receptor (PTCH1), a crucial component of the Sonic Hedgehog (SHH) pathway responsible for various developmental processes, including ventralization patterning, proliferation, and differentiation of neuronal precursor cells (Ingham, 2022; Carballo et al., 2018). Nevertheless, PTCHD1 does not functionally substitute PTCH1 in canonical Sonic Hedgehog signaling, as it neither represses Smoothened nor binds Sonic Hedgehog (Ung et al., 2018; Tora et al., 2017), but it retains the ability to bind cholesterol similarly to PTCH1 (Hiltunen et al., 2023). PTCHD1 protein contributes to the cholesterol content regulation and trafficking of μ-opioid receptors, revealing unexpected connections to addiction processes and cholesterol signaling pathways (Maza et al., 2022).

In fact, it is well established that PTCHD1 acts as a postsynaptic membrane protein and interacts with the postsynaptic density proteome and notably with the major scaffolding proteins PSD95 and SAP102 via its PDZ binding motif of the C-terminal intracellular tail (Tora et al., 2017; Ung et al., 2018). In addition, PTCHD1 is involved in inflammatory signaling pathway (Murakami et al., 2019). Nevertheless, the precise roles for PTCHD1 in brain development and maturation need to be determined.

Transcriptomic analyses have revealed expression of *PTCHD1* in most brain regions in primates and in adult mice, particularly in the cerebellar, cortical, and hypothalamic structures (Bakken et al., 2015; Wells et al., 2016; Tora et al., 2017; Ung et al., 2018). During postnatal development, a distinct peak in the Thalamic Reticular Nucleus (TRN) is observed from birth to postnatal day 14 (P14) in wildtype mice (Wells et al., 2016), and deletion of *Ptchd1* leads to attenuation of TRN activity, manifesting as hyperactivity and attention deficit behavioral effects in *Ptchd1* knockout mice. Furthermore, *Ptchd1* deficiency significantly impacts the structural maturation and organization of excitatory synapses in mice, resulting in cognitive dysfunction, motor disabilities and hyperactivity in *Ptchd1* knockout models (Wells et al., 2016; Tora et al., 2017; Ung et al., 2018).

Recent developed human-derived induced pluripotent stem cells (hiPSCs) and organoid models have provided valuable insights into PTCHD1-associated neurodevelopmental dysfunctions. Patient-derived hiPSCs with *PTCHD1* deletions demonstrated impaired synaptic function, including reduced excitatory synaptic transmission (Ross et al., 2020; Farley et al., 2024), mirroring findings obtained from *Ptchd1* knockout mice (Ung et al., 2018). Furthermore, ventral thalamic organoid models with *PTCHD1* alterations revealed deficits in neuronal function, particularly within the thalamic reticular nucleus (TRN), emphasizing the critical role of PTCHD1 in neural circuit development and synaptic plasticity (Kiral et al., 2023).

Despite the established association between *PTCHD1* deletions in patients and the NDD phenotype, recent investigations into *Ptchd1* dysfunctions in mice have predominantly focused on its neuronal and synaptic roles at the adult stage, neglecting an exploration of the precise role of PTCHD1 across the brain development.

## 2. *PTCHD1* expression in mouse brain development

*Ptchd1* gene is expressed early during brain development, at embryonic stages 9 (E9) and E14, in the whole embryo and CNS, respectively. Real-time qPCR analyses covering E13 to postnatal day 35 (P35; Ung et al., 2018) and P5 to P60 (Tora et al., 2017) illustrated an increase in *Ptchd1* expression from embryonic E13, peaking at E18-P7 across most brain regions. Notably, mRNA *in situ* hybridization at P0 highlighted restricted expression in the thalamic reticular nucleus (TRN), particularly heightened in the thalamus at P5 and persisting until P15 (Wells et al., 2016; Tora et al., 2017). Further investigations at P12 emphasized the presence of *Ptchd1* in the TRN, cortex, striatum, and notably in the cerebellum, spanning external germinal layer cells, Purkinje cell layer, and internal cell layer (Tora et al., 2017). At the adult stage, *Ptchd1* transcripts were identified in the TRN, cortex, striatum, cerebellum, specifically in the internal granule cell layer, and in the dentate gyrus of the hippocampus at P21 and P60. Recent findings utilizing the FISH technology revealed increasing *Ptchd1* mRNA expression in the anterodorsal thalamus (Roy et al., 2021). Investigations in murine models further support and extend these findings, demonstrating developmentally regulated *Ptchd1* expression across embryonic and postnatal stages, particularly in the thalamus and cerebellum. Together, these findings underscore the critical role of PTCHD1 in the CNS development, offering insights into its potential contributions to neuronal process and circuit formation.

## 3. PTCHD1 in the PATCHED family

Due to its structural resemblance to the PTCH1, PTCHD1 was initially hypothesized to function as another PATCHED transmembrane receptor, would act as a key regulator of the Sonic Hedgehog (SHH) signaling pathway and may be essential for neurodevelopmental processes like neuronal precursor proliferation, differentiation, and ventral patterning (Carballo et al., 2018; Fig. 1). However, unlike PTCH1, PTCHD1 does not bind SHH nor participate in the canonical SHH signaling pathway, distinguishing it from other Patched receptors (Ung et al., 2018; Hiltunen et al., 2023). Intriguingly, PTCHD4, another member of the PTCHD subfamily, also does not interact with SHH, further suggesting functional divergence within the PATCHED family.

Members of the PATCHED family, including PTCH, PTCHD, and PTR (Patched-related) proteins, play also vital roles in the transport of steols, sterol-modified proteins, and lipids (Kuwabara and Labouesse, 2002). They encompass diverse subtypes, including Patched (PTC/PTCH) proteins, Patched-related (PTR) proteins, human Niemann-Pick C disease (NPC1 and NPC1L1) proteins, Dispatched (DISP) proteins (DISP1, DISP2), and Patched domain-containing (PTCHD) proteins, as delineated in Fig. 1 (Pastore et al., 2022). Interestingly, a shared structural framework characterized by the presence of a Sterol-Sensing Domain (SSD, blue domain in Fig. 1) has been identified in the PATCHED Homolog Domain (PTCHD) subfamily, which includes PTCHD1, PTCHD2/DISP3, PTCHD3, and PTCHD4/PTCH53.

Recent findings have demonstrated the ability of PTCHD1 to bind cholesterol and regulate membrane cholesterol content, suggesting a novel functional role for the protein (Fig. 1). This function, closely related to PTR transporters and RND (resistance-nodulation-division) motif transporters, underscores the significance of PTCHD1's sterolsensing domain in mediating cholesterol-related processes, distinct from the role of PTCHD2/DISP3. Additionally, Hiltunen and collaborators have reported that AlphaFold2 predictions of PTCHD1 reveal structural homology with several cholesterol transporters within the PATCHED domain-containing family, notably Dispatched 1 (DISP1), NPC1, and PTCH1; but comparative sequence alignments differ (Hiltunen et al., 2023), as do PTCHD1-specific interacting partners (see Sections 6.2–6.4).

Despite their commonality, members of the PTCHD family show distinct subcellular localization, expression patterns, and functions
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Comprehensive analysis of PTCH family, with PTCHD subfamily proteins and their link with PTCHD1 and contribution to brain developmental processes and diseases.; The Patched (Ptch) receptor functions as a key negative regulator of the Hedgehog signaling pathway by inhibiting the activity of the transmembrane protein Smoothened (Smo) in the absence of the Sonic Hedgehog (Shh) ligand. Upon Shh binding, Ptch undergoes a conformational change that relieves its inhibition of Smo, thereby enabling the activation of downstream signaling cascades essential for regulating cellular proliferation, differentiation, and morphogenesis (indicated by the green arrow). PTCHD1 and PTCHD4 do not participate in the canonical SHH signaling pathway. NPC1 (and NPC1L1), DISP1, DISP2, DISP3/PTCHD2, and PTCHD1 are implicated in the cholesterol regulation pathway (Blue domain are for SSD sterol sensing domain; see Section 6.2) (indicated by the black arrow). PTCHD2/DISP3 plays a role in the neurogenesis process (purple dash arrow). PTCHD1 is involved in synaptic regulation (red arrow). What about a role for PTCHD1 during neurogenesis process (Arrow question mark). Biorender was used for the schema design. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

(Zhong et al., 2014). They also differ in their intracellular C-terminal domain (CTD), a region involved in protein-protein interaction and important for trafficking, and to trigger downstream signaling pathways. Although PTCHD1 and PTCHD4 share a PDZ binding motif, they are involved in separate functional pathways (Fig. 1). Of note, PTCHD3 is specifically expressed in male germ cells and deviates from the typical structure of the PTCHD subfamily (Fig. 1; Ghahramani Seno et al., 2011). Notably, PTCHD3 lacks the second cluster of five transmembrane domains, as reported by Fan and collaborators (Fan et al., 2007). Furthermore, PTCHD4 is transcriptionally activated by the tumor suppressor protein p53 (Fig. 1; Chung et al., 2014).

## 4. PTCHD1 secondary protein structure, subcellular localization and post-translational modifications

The subcellular localization, stabilization, and trafficking of PTCHD1 have been investigated in recent studies and in the next subsections, we highlight (Fig. 2) the potential and functional roles of various motifs and sequences identified in the PTCHD1 protein sequence.

### 4.1. The secondary structure of PTCHD1 predicts a transmembrane receptor

PTCHD1 gene located on short arm of X chromosome on XP22.11 covered nearly 70 Kb. The larger transcript (NM_173495.3) spanning

about 13 Kb contains three exons. The full-length PTCHD1 protein (Uniprot AF_Q96NR3) consists of 888 amino acids and exhibits a predicted secondary structure similar to the PTCH membrane receptor (Kuwabara and Labouesse, 2002). This similarity arises from a common PATCHED family domain, characteristic of PATCHED family proteins, including PTCHD1. These proteins share several transmembrane domains (See detailed image from Pymol software, Fig. 2A). Notably, five of these TMD form a sterol-sensing domain (SSD) spanning approximately 180 amino acids, a structural motif well-conserved and largely documented in the literature (Carstea et al., 1997; Zhong et al., 2014; Fig. 2B). PTCHD1 structural analysis reveals that both N- and C-terminal regions are intracellular. The C-terminal domain (CTD) of PTCHD1 bears distinctive regions including a charged amino acid domain and a distal PDZ-binding motif, potentially facilitating specific protein-protein interactions and downstream signaling modulation (Fig. 2). Notably, the C-terminal PDZ-binding motif ITTV presenting the consensus sequence X-T-X-V, common in synaptic proteins like EXOC4 and NLGN, underscores the potential involvement of PTCHD1 in the postsynaptic scaffold interactome (Sans et al., 2003; Meyer et al., 2004; Zhu et al., 2016, Ung et al., 2018).

### 4.2. PTCHD1 localization at neuronal synapses

Noor et al. initially reported the plasma membrane localization of PTCHD1-GFP fusion protein in COS7, SK-N-SH and Neuroblastoma
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Structural visualization and schematic representation of PTCHD1 protein structure: Predicted structural domains, Missense mutation sites linked with NDDs and predicted post-translational modification sites. A. Using the NCBI PTCHD1 human protein sequence (NP_775766.2) in FASTA format, the structure was predicted via AlphaFold2 and converted into a PDB file. The resulting 3D model was then visualized and analyzed using PyMOL to examine its structural features. B. Predicted structural domains of PTCHD1, as described by Halewa et al. (2021), featuring 12 transmembrane domains with two extracellular loops and a sterol-sensing domain (SSD, TM 2–6). The protein possesses intracellular N- and C-terminal ends, with the PDZ-binding domain (ITTV sequence) highlighted in a red rectangle. Missense mutation sites associated with neurodevelopmental disorders (NDDs) are indicated by orange squares, corresponding to missense variants listed in Table 3. Predicted post-translational modifications (PTM) sites are marked by star symbols, with green indicating N-glycosylation sites, violet representing predicted acetylation sites, blue denoting predicted phosphorylation sites, as detailed in Table 1. Biorender was used for the schema design. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

(N2A) cell lines as well as in primary neuronal cultures where PTCHD1 is particularly enriched within PSD95-labeled post-synaptic sites (Noor et al., 2010; Ung et al., 2018; Halewa et al., 2021). While specific details regarding the trafficking of PTCHD1 remain limited, evidence suggests that the C-terminal amino acid stretch ranging from 850 to 873 is crucial for its synaptic targeting (Fig. 2B). Surprisingly, this synaptic addressing is not dependent on the distal PDZ binding motif of PTCHD1 in contrast to several other synaptic proteins such as EXOC4 and SYNGAP1 (Ung et al., 2018; Walkup et al., 2015). Interestingly, Ascano et al. used PAR-CLIP and RNA sequencing to identify FMRP targets, revealing a potential interaction with PTCHD1 mRNA. This finding implies that FMRP regulates the local synaptic translation of PTCHD1 mRNA in response to mGluR activation (Ascano et al., 2012).

### 4.3. Post-translational modifications of PTCHD1

PTCHD1 is covalently modified by different post-translational modifications (PTM), which may impact its degradation, subcellular localization, trafficking and/or function (Fig. 2B). Several predicted PTM, including N-glycosylation, phosphorylation, acetylation, ubiquitination and SUMOylation can be identified in PTCHD1 using prediction software (Table 1; Fig. 2B).

Xie et al. (2024) identified seven predicted glycosylation sites and demonstrated that several candidate PTCHD1 variants associated with NDD were found to significantly alter stability and glycosylation status of the respective mutated PTCHD1 proteins (Fig. 2B). Additionally, a photo-click cholesterol assay revealed specific cholesterol binding to both the native and glycosylated forms of PTCHD1, suggesting a potential regulatory role of the protein in lipid interactions (Fig. 2B; Hiltunen et al., 2023). As cholesterol is crucial for synaptic transmitter localization and function, mutations in PTCHD1 may disrupt synaptic signaling by impairing cholesterol transport in dendritic spines, without impacting scaffolding proteins interactions (Xie et al., 2024).

Phosphorylation of PTCHD1 is predicted to occur after its trafficking to the plasma membrane, where it may play a role in modulating protein-protein interactions and downstream signaling pathways

<caption>**Table 1**</caption>

PTCHD1 predicted post translational modifications (PTM) using NetNGlyc - 1.0 and MusiteDeep software.

<table><thead><tr><th>Predicted affected region of the protein</th><th>Site</th><th>predicted PTM</th><th>BDD (update January 2025)</th><th>Potential N Glyc (threshold = 0.5)</th></tr></thead><tbody><tr><td rowspan="4">Extracellular loop 1</td><td>N77</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.7116</td></tr><tr><td>N133</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.6072</td></tr><tr><td>N167</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.7630</td></tr><tr><td>N319</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.6223</td></tr><tr><td rowspan="2">Extracellular</td><td>N326</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.5251</td></tr><tr><td>K353</td><td>Ubiquitination</td><td>MusiteDeep</td><td></td></tr><tr><td rowspan="5">Intracellular</td><td>K446</td><td>SUMOylation</td><td>MusiteDeep</td><td></td></tr><tr><td>K453</td><td>Ubiquitination</td><td>MusiteDeep</td><td></td></tr><tr><td>S466</td><td>Phosphorylation</td><td>MusiteDeep</td><td></td></tr><tr><td>N551</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.1230</td></tr><tr><td>N568</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.5549</td></tr><tr><td rowspan="4">Extracellular loop 2</td><td>K597</td><td>Acetylation</td><td>Phosphosite</td><td></td></tr><tr><td>N599</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.4989</td></tr><tr><td>N608</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.4946</td></tr><tr><td>N683</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.4346</td></tr><tr><td>Extracellular</td><td>N818</td><td>N-glycosylation</td><td>NetNGlyc - 1.0</td><td>0.6641</td></tr><tr><td>Intracellular</td><td>S852</td><td>Phosphorylation</td><td>MusiteDeep</td><td></td></tr></tbody></table>

(Fig. 2B). Two serine residues located in the third intracellular loop (S466) and in the C-terminal part of PTCHD1 (S852) are predicted to be phosphorylated using the post-translational modification site prediction MusiteDeep solution (Wang et al., 2017, 2019, 2020).

PTCHD1 may also be targeted for either ubiquitination or SUMOylation, two PTMs leading to the covalent modification of the lateral chain of specific lysine residues by the Ubiquitin (K353, K453) or the Small Ubiquitin Modifying (SUMO) enzymatic system, respectively. MusiteDeep software prediction indicated that two lysine residues in the second (K353) and third intracellular loops (K453) may be ubiquitinated, leaving the exciting possibility that ubiquitination may participate in the regulation of PTCHD1 stability (Fig. 2B). Prediction software also highlights the lysine residue K446 within the third intracellular loop
as a potential SUMOylation site for PTCHD1 (Fig. 2B). Since SUMOylation is known to regulate protein-protein interactions and the trafficking of pre- and post-synaptic proteins (Chato-Astrain et al., 2024), it would definitely be of interest to assess the potential role of PTCHD1 SUMOylation in its synaptic function. However, the precise mechanisms by which these PTMs may influence PTCHD1 function, stability and trafficking remain primarily unknown. Therefore, further studies are now needed to elucidate the functional consequences of these predicted PTMs and how pathological missense mutations may alter the synaptic function of PTCHD1 by impacting these protein modifications.

Interestingly, functional studies have shown that mutations in *PTCHD1* associated with ID-ASD disrupt protein stability, expression, and subcellular localization (Halewa et al., 2021). Among 13 missense variants identified in affected individuals, 6 particularly affect the transmembrane domains and exhibited reduced protein expression, likely due to ER retention (Fig. 2B). Since additional *PTCHD1* variants continue to be identified (Pastore et al., 2022; Pastore et al., 2024, Farley et al., 2024; Montanaro et al., 2024), it remains essential to incorporate PTCHD1 PTM analysis in line with *PTCHD1* missense variants to understand its regulation sites.

# 5. PTCHD1: a central role at synapses?

Despite its structural resemblance to Patched receptors, PTCHD1 operates independently of the canonical Sonic Hedgehog (SHH) pathway. Instead, it plays a distinct role in cholesterol binding and regulation, positioning PTCHD1 as a key sterol-sensing protein. *Ptchd1* exhibits dynamic and region-specific expression patterns throughout mouse brain development, with critical roles in synaptic function and neuronal activity. During early embryogenesis, *Ptchd1* is expressed broadly across the developing mouse brain starting at embryonic day 9 (E9), with increasing levels from E13 to E18. Peak expression reaches highest levels by E18-P7 in most brain regions, coinciding with dendritogenesis and early synaptogenesis. Then, PTCHD1 is mainly localized at synapses, indicating that it may exert a central role in neurodevelopmental processes and brain communication. Despite recent discoveries highlighting PTCHD1 as a synaptic membrane protein interacting with postsynaptic density proteins and components of the retromer complex, its specific role in synaptogenesis is still not well understood. In the following sections, we reviewed findings from the past two decades to clarify the involvement of PTCHD1 in these critical brain developmental and maturation processes.

## 5.1. Involvement of PTCHD1 in synaptogenesis

### 5.1.1. PTCHD1 and expression of immediate-early genes

The brain development is intricately regulated by activity-dependent activation of immediate-early genes (Kim et al., 2018). Among immediate-early genes, *NPAS4* and *Egr1* are particularly well-described for their implication in NDDs. *NPAS4*, a calcium-responsive transcription factor, plays a pivotal role in orchestrating the excitatory-inhibitory balance which is crucial for the establishment and plasticity of neural circuits (Fu et al., 2020) that are often disturbed in ASD (Golden et al., 2018). Recent studies have elucidated the association of *NPAS4* with *NuA4* complexes at synapses, emphasizing its significance, particularly given the implications of mutations in *NuA4* complex genes in NDDs (Pollina et al., 2023). Additionally, EGR1 emerges as a key regulator of synaptic plasticity and neuronal activity, further underscoring the intricate regulatory network governing synaptic development (Duclot, Kabbaj, 2017). In addition, previous investigation has also revealed a 50% increase in the expression of the immediate-early genes *Egr1* and *Npas4* in the hippocampus of *Ptchd1* knockout mice beginning at the postnatal day 30 (Ung et al., 2018) further highlighting the importance of this pathway in the developing brain.

### 5.1.2. PTCHD1 role in synapse formation

Evidence regarding the role of PTCHD1 in synaptogenesis came from its spatiotemporal expression pattern, which mirrors the early stages of synaptogenesis in mice, and its protein localization within synaptic fractions in mouse brains further emphasizing its involvement in synaptic development (Tora et al., 2017; Ung et al., 2018). In addition, synapse formation is intricately linked to the activation of potassium and calcium channels, as evidenced by the upregulation of *Ptchd1* transcript levels in cortical primary neurons following depolarization induced by KCl stimulation (Kim et al., 2010). Moreover, altered intracellular calcium dynamics were measured in *Ptchd1* knockout mouse neurons, revealing a potential role for PTCHD1 in modulating intracellular calcium signaling pathways (Wells et al., 2016). Furthermore, assessment of synapse formation in *Ptchd1* knockout mouse models revealed structural deficits at excitatory synapses (Fig. 4D), characterized by a significant reduction in excitatory synapse number and ultrastructural abnormalities such as decreased postsynaptic density (PSD) and synaptic cleft width, along with the accumulation of neurotransmitter vesicles (Ung et al., 2018; Tora et al., 2017). Intriguingly, while dendritic spine density remained unchanged in dentate granule cells from adult *Ptchd1* knockout mice, alterations in synaptic architecture and function were evident, highlighting the multifaceted roles of PTCHD1 in synaptogenesis processes.

## 5.2. Major roles of PTCHD1 in synaptic architecture and neuronal transport

Although the precise role of PTCHD1 as a receptor is not fully understood, its interactions with key postsynaptic scaffolding proteins such as PSD95, DLG3 (SAP102), and SNX27 suggest a significant regulatory function within the synapse (Table 2, Fig. 3; Ung et al., 2018; Tora et al., 2017). Therefore, understanding the synaptic role of PTCHD1 requires a comprehensive analysis of its interacting partners and associated signaling cascades.

### 5.2.1. PTCHD1 in postsynaptic density scaffolding

The postsynaptic density (PSD) is essential for synapse organization and maintenance (Laumonnier et al., 2007). Its composition in neurotransmitter receptors usually defines the synapse specificity. The intracellular C-terminal domain of PTCHD1 is implicated in binding critical postsynaptic scaffolding proteins via its PDZ-binding motif (Zhu et al., 2016; Tora et al., 2017; Ung et al., 2018). Experimental evidence from GST (*Glutathione-S-Transferase*) pull-down assays using synaptoneurosomal fractions from cortices and total brain lysates demonstrates the association of PTCHD1 with several important PSD proteins, including PSD95 and SAP102, members of the MAGUKs family (Table 2, Fig. 3; Tora et al., 2017; Ung et al., 2018, Chen et al., 2015). Recent investigations have identified interactions with SAP102b, involving the third PDZ domain of SAP102 and SH3 (SRC homology 3) and GK (Guanylate Kinase) domains (Xie et al., 2024). PTCHD1 expands its PSD-interacting network by engaging additional components (Fig. 3) such as DLG1, DLG2, MAGI1, MAGI3, and VELI1 via their PDZ-binding motifs (Tora et al., 2017). These interactions are pivotal for the structural integrity of glutamatergic synapses, allowing the tethering of N-methyl-D-aspartate (NMDA) and AMPA receptors at synapses (Laumonnier et al., 2007; Sahin and Sur, 2015). Additional evidence arises from ultrastructural analyses of excitatory synapses in the *Ptchd1* knockout model, where the absence of PTCHD1 expression significantly destabilizes the PSD architecture (Ung et al., 2018).

### 5.2.2. PTCHD1 in neuronal transport

The unavailability of a specific antibody for PTCHD1 is a clear limitation in identifying its potential presynaptic localization, although recent findings suggest a potential role of the protein at presynaptic sites due to its interacting partners (Pastore et al., 2024). The intracellular C-terminal tail of PTCHD1 interacts with the Sorting Nexin 27 (Snx-27),
<table><caption>**Table 2**
PTCHD1 interacting partners mapped to neuronal and synaptic pathways and functions.</caption><thead><tr><th>General function</th><th>Molecular function</th><th>Synaptic localization</th><th>Interacting partners</th><th>Via ITTV sequence</th><th>Via C-terminal tail</th><th>Full length PTCHD1 fused with GFP</th><th>Name</th><th>Validation: method and lysate</th></tr></thead><tbody><tr><td rowspan="10">Scaffolding proteins from PSD</td><td rowspan="10">Post synaptic density scaffolding</td><td rowspan="10">Scaffolding proteins</td><td>PSD95<sup>s</sup></td><td></td><td></td><td></td><td>Postsynaptic Density Protein 95</td><td>**Cx/**Syn. Cx/*Cx</td></tr><tr><td>SAP102</td><td></td><td></td><td></td><td>Synapse Associated Protein 102</td><td>***H/**Syn. Cx/*Cx</td></tr><tr><td>MAGI1</td><td></td><td></td><td></td><td>Membrane Associated Guanylate Kinase, WW And PDZ Domain Containing 1</td><td>*Cx</td></tr><tr><td>MAGI3</td><td></td><td></td><td></td><td>Membrane Associated Guanylate Kinase, WW And PDZ Domain Containing 3</td><td>*Cx</td></tr><tr><td>DLG1<sup>s</sup></td><td></td><td></td><td></td><td>Discs Large MAGUK Scaffold Protein 1</td><td>*Cx</td></tr><tr><td>DLG2<sup>s</sup></td><td></td><td></td><td></td><td>Discs Large MAGUK Scaffold Protein 2</td><td>*Cx</td></tr><tr><td>MPP2</td><td></td><td></td><td></td><td>MAGUK p55 subfamily member 2</td><td>*Cx</td></tr><tr><td>MPP6/ VAM1</td><td></td><td></td><td></td><td>MAGUK p55 subfamily member 6 or PALS2</td><td>*Cx</td></tr><tr><td>SNTA1</td><td></td><td></td><td></td><td>Syntrophin (dystrophin-associated protein complex)</td><td>*Cx</td></tr><tr><td>CSKP</td><td></td><td></td><td></td><td>Protein Peripheral plasma membrane protein</td><td>*Cx</td></tr><tr><td rowspan="6">Intracellular protein trafficking and transport</td><td>Trans-Golgi network transport vesicle</td><td>Trans-Golgi network transport vesicle</td><td></td><td></td><td></td><td></td><td>Golgi-associated PDZ and coiled-coil motif-containing protein</td><td>*Cx</td></tr><tr><td rowspan="2">Microtubule transport Retromer complex</td><td rowspan="2">Dynein complex</td><td>Pre</td><td></td><td>DCTN3</td><td></td><td>Dynactin subunit 3</td><td>*Cx</td></tr><tr><td>Pre</td><td></td><td>VPS35</td><td></td><td>VPS35 Retromer Complex Component</td><td>**Cx/*Cx</td></tr><tr><td rowspan="2">Lysosome membrane Nuclear pore complex</td><td rowspan="2">Transporter</td><td>Pre</td><td></td><td>VPS26B</td><td></td><td>VPS26 Retromer Complex Component B</td><td>*Cx</td></tr><tr><td>Pre</td><td></td><td>SNX27</td><td></td><td>Sorting Nexin 27</td><td>*Cx</td></tr><tr><td rowspan="4">Cell polarity and adhesion</td><td rowspan="4">Membrane asymmetry</td><td rowspan="4">asymmetric distribution of channels and receptors at the plasma membrane of polarized cells</td><td>Pre</td><td></td><td>GRN</td><td></td><td>Progranulin</td><td>*Cx</td></tr><tr><td>Pre</td><td></td><td>IMA3</td><td></td><td>Importin Alpha 3</td><td>*Cx</td></tr><tr><td></td><td></td><td>VIAAT (VGAT)</td><td></td><td>Vesicular GABA Transporter</td><td>*Cx</td></tr><tr><td></td><td></td><td></td><td></td><td>Lin-7 Homolog A, Crumbs Cell Polarity Complex Component</td><td>*Cx</td></tr><tr><td rowspan="5">Translation</td><td rowspan="5">Translation (Ribosomal subunit)</td><td rowspan="5">Ribosomal Protein</td><td>Pre</td><td></td><td>LIN7A</td><td></td><td>Lin-7 Homolog C, Crumbs Cell Polarity Complex Component</td><td>*Cx</td></tr><tr><td>Pre</td><td></td><td>LIN7C</td><td></td><td>60S ribosomal protein L5</td><td>*Cx</td></tr><tr><td></td><td></td><td>RL5, 7, 8, 18A, 27, 31, 32, 37</td><td></td><td>Ribosomal Protein S26</td><td>*Cx</td></tr><tr><td></td><td></td><td>RS26</td><td></td><td>Ribosomal Protein L31</td><td>*Cx</td></tr><tr><td></td><td></td><td>RPL31</td><td></td><td>Ribosomal Protein L31 Mitochondrial</td><td>*Cx</td></tr><tr><td></td><td></td><td></td><td></td><td>RT31</td><td></td><td>Ribosomal Protein S31</td><td>*Cx</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>General function</th><th>Molecular function</th><th>Synaptic localization</th><th>Interacting partners</th><th>Via ITTV sequence</th><th>Via C-terminal tail</th><th>Full length PTCHD1 fused with GFP</th><th>Name</th><th>Validation: method and lysate</th></tr></thead><tbody><tr><td rowspan="10">Transcription and signalling pathway</td><td rowspan="4">RNA stress granules</td><td>RNA binding/regulation of stress granule and P-body formation</td><td rowspan="4"></td><td rowspan="4"></td><td rowspan="4"></td><td>ATXN2L</td><td>Ataxin-2-like protein</td><td>**H</td></tr><tr><td>RNA binding</td><td>NUFIP2</td><td>FMR1-interacting protein NUFIP2</td><td>**H</td></tr><tr><td rowspan="2">RNA helicase</td><td>PABP1</td><td>Polyadenylate-binding protein 1</td><td>**H</td></tr><tr><td>DDX3X<sup>S</sup></td><td>ATP-dependent RNA helicase DDX3X</td><td>**H</td></tr><tr><td rowspan="2">Scaffold protein/ RNA stress granules</td><td>DDX6<sup>S</sup></td><td>Probable ATP-dependent RNA helicase DDX6</td><td>**H</td></tr><tr><td>G3BP2<sup>S</sup></td><td>Ras GTPase-activating protein-binding protein 2</td><td>**H</td></tr><tr><td rowspan="5">Regulation</td><td>Kinase</td><td rowspan="5">pre</td><td rowspan="5"></td><td rowspan="5"></td><td>Dual specificity tyrosine-phosphorylation-regulated kinase 1A</td><td>**H</td></tr><tr><td>Phosphatase</td><td>Protein Phosphatase 2</td><td>*Cx</td></tr><tr><td>Perinuclear region of cytoplasm</td><td>Regulatory Subunit B alpha</td><td>*Cx</td></tr><tr><td>Chaperone/ protein quality control system / mitochondrial import/ ER-associated degradation</td><td>Cell division cycle and apoptosis regulator protein 1</td><td></td></tr><tr><td>Nucleosomes</td><td>Heat shock cognate 71 kDa protein</td><td>**H</td></tr><tr><td rowspan="4">Actin regulation</td><td>actinin binding</td><td rowspan="4"></td><td rowspan="4"></td><td rowspan="4"></td><td>H10</td><td>Heat shock protein beta-1</td><td>*Cx</td></tr><tr><td rowspan="3">Post-translational modification / ubiquitination</td><td>Post-translational modification</td><td>H11</td><td>Histone H1.0</td><td>*Cx</td></tr><tr><td rowspan="2">Post-translational modification</td><td>Post-translational modification</td><td>H12</td><td>Histone H1.1</td><td>*Cx</td></tr><tr><td>mitochondrial protein synthesis / Required for enhanced channeling of cholesterol for hormone-dependent steroidogenesis</td><td>mitochondrial inner membrane and nucleoids</td><td>CSRP1</td><td>Cysteine and glycine-rich protein 1</td><td>**H</td></tr><tr><td rowspan="2">Post-translational modification</td><td>mitochondrial protein synthesis / Required for enhanced channeling of cholesterol for hormone-dependent steroidogenesis</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td>DDB1- and CUL4-associated factor 7</td><td>DDB1- and CUL4-associated factor 7</td><td>**H</td></tr><tr><td>mitochondrial inner membrane and nucleoids</td><td>UBB</td><td>Polyubiquitin-B</td><td>**H</td></tr><tr><td>Cholesterol pathway</td><td>mitochondrial inner membrane and nucleoids</td><td>mitochondrial inner membrane and nucleoids</td><td>ATAD3A<sup>O</sup></td><td>ATPase family AAA domain-containing protein 3A</td><td>**H</td></tr></tbody></table>

VPS26B, and VPS35 *in vitro*, and these interactions occurs independently of its PDZ-binding motif (Tora et al., 2017), establishing a link between PTCHD1 and the retromer complex (Wang, Bellen, 2015; Choy et al., 2014; Brodin and Shupliakov, 2018). The retromer complex comprises several Sorting Nexins including SNX1, SNX2, VPS35, VPS29, and VPS26B. They are essential to the trafficking of transmembrane proteins from endosomes to the trans-Golgi network (TGN) or the plasma membrane (Fig. 3; Wang, Bellen, 2015). Furthermore, PTCHD1 interacts though its first extracellular luminal loop with SNAPIN (Fig. 3), a protein involved in the docking and fusion of synaptic vesicles, in the biogenesis of lysosome-related organelles complex 1 (BLOC-1) and synaptic transmission (Pastore et al., 2025, Ilardi et al., 1999). This interaction of PTCHD1 with the retromer complex may reflect a role of PTCHD1 in the endosomal-lysosomal trafficking system, thereby

influencing synaptic vesicle dynamics. This could therefore explain the accumulation of presynaptic vesicles observed in the *Ptchd1*<sup>-/-</sup> knockout hippocampal presynaptic site of excitatory synapse and its impact on the paired pulse ratio on CA1 pyramidal neurons observed via electrophysiological analysis on coronal slices (Ung et al., 2018). Interestingly, NPC1 is an intracellular cholesterol transporter active in late endosomes and lysosomes, mediating the export of unesterified cholesterol (Yu et al., 2014). Together, the data likely reflect a disruption in synaptic vesicle dynamics due to PTCHD1's role in the endosomal-lysosomal system. Further investigation is needed to clarify whether this is linked to defects in cholesterol homeostasis, which is crucial for membrane fluidity and synaptic function and regarding notably recent publication of Maza and colleagues (See section 6.4.2) (Maza et al., 2022).

<footnote>*: GST + Mass-spectrometry; **: GST + Immunoblotting; ***: Immunoprecipitation; #: PAR-CLIP + RNA seq; H: HEK293T; Cx: Cortex.</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** PTCHD1-interacting partners at excitatory synapses.; PTCHD1 plays a crucial role in postsynaptic density (PSD) organization by interacting with key scaffolding proteins via its PDZ-binding motif. Experimental evidence demonstrates its association with PSD-95, SAP102, and other MAGUK family members, contributing to glutamatergic synapse integrity by tethering NMDA and AMPA receptors. PTCHD1 also interacts with proteins like Dlg1, Dlg2, Magi1, Magi3, and Veli1, further expanding its PSD network. While PTCHD1 interacts with presynaptic proteins such as Sorting Nexin 27 (Snx-27), VPS26B, VPS35 (retromer complex), and SNAPIN (t-SNARE complex), its precise localization at the presynaptic site remains unclear. Further investigations using specific antibodies or super-resolution imaging are needed to determine its presynaptic presence and function. Biorender was used for the design.</figcaption></figure>

### 5.3. *PTCHD1* in synaptic plasticity and activity

It was previously defined that FMRP regulates the local synaptic translation of PTCHD1 mRNA in response to mGluR activation (Ascano et al., 2012), emphasizing its reactive role in synaptic plasticity. Studies on *Ptchd1* knockout mice have revealed alterations in synaptic transmission and activity, indicative of its involvement in regulating neuronal circuitry. Specifically, *Ptchd1*⁻/γ mice exhibit a significant decrease in the frequency of excitatory postsynaptic currents (EPSCs) in hippocampal CA1 pyramidal neurons, along with alterations in the releasing probability of excitatory synapses onto these neurons (Ung et al., 2018). Additionally, dentate granule cells lacking *PTCHD1* display changes in the excitation/inhibition ratio and increased frequency of spontaneous excitatory and inhibitory events, highlighting the role of PTCHD1 in modulating synaptic activity (Tora et al., 2017). The observed excitatory-inhibitory imbalance in hippocampal neuronal circuits of *Ptchd1*⁻/γ mice underscores the significance of PTCHD1 in synaptic function and plasticity. Moreover, the altered SK currents in TRN neurons lacking *Ptchd1* further emphasize its role in regulating neuronal excitability (Wells et al., 2016). However, contradictory findings regarding SK2 channel accumulation in human thalamic organoids (Kiral et al., 2023) suggest the complexity of PTCHD1 impact on synaptic function and the need for further investigations.

In summary, the above-mentioned findings highlight PTCHD1 as a key player in neuronal and synaptic trafficking, with its interactions linking the protein to critical pathways such as the retromer complex and endosomal-lysosomal systems. The possible presynaptic localization of PTCHD1, coupled with its influence on synaptic vesicle cycling and cholesterol transport, suggests a broader role in maintaining neuronal homeostasis and synaptic function. This positions PTCHD1 as a mediator

of neuronal trafficking, with far reaching implications regarding its roles in the etiology of NDDs. Future studies using specific PTCHD1 antibodies associated with super-resolution imaging are now needed to unravel the precise mechanisms of action of PTCHD1 and neurological relevance.

## 6. PTCHD1 as a regulator of major signaling pathways

Here we explore emerging data regarding the intricate interaction and signaling networks surrounding PTCHD1. Indeed, recent exciting studies revealed novel insights on the involvement of PTCHD1 in RNA translation as well as in cholesterol and inflammatory signaling pathways.

### 6.1. *PTCHD1* role in RNA translation regulation?

Previous findings linked PTCHD1 regulation by FMRP emphasizing its multifaceted involvement in neuronal cellular processes (Ascano et al., 2012; Chen et al., 2014). Using non neuronal cell line HEK293, Hiltunen and collaborators recently reported *in vitro* interactions of PTCHD1 with 13 candidate proteins including ribonucleoproteins and stress granule proteins (Table 2; Hiltunen et al., 2023), such as DDX3X (Lennox et al., 2020; Snijders Blok et al., 2015) and DYRK1A (two proteins associated with NDDs) (Atas-Ozcan et al., 2021), or NUFIP2, PABP1, DDX6, and G3BP2 (Hiltunen et al., 2023) (Fig. 3). Ribonucleoproteins are essential for local translation in axons and dendrites as well as in synaptic sites, allowing fast responses to neuronal activation and synaptic plasticity. These findings suggest a role of PTCHD1 in regulating RNA transport and translation by interacting with ribonucleoproteins and stress granule proteins, including key neurodevelopmental
disorder-associated factors like *DDX3X* and *DYRK1A*. Nevertheless, it is unclear whether *PTCHD1* transmembrane receptor does interact endogenously with those proteins, directly or indirectly, through specific molecular complexes.

## 6.2. *PTCHD1* in the cholesterol content regulation

The secondary structure of *PTCHD1* includes a Sterol Sensing Domain (SSD) with five transmembrane domains responsible for sterol regulation (see Section 3), such as members of the Patched family, including *PTCH*, *PTCHD*, and *PTR* proteins (Kuwabara and Labouesse, 2002). In fact, a recent study from Maza and collaborators revealed that *PTCHD1* controls μ-opioid receptor (MOR) trafficking through membrane cholesterol content regulation (Maza et al., 2022). The loss of *Ptch1* reduces opioid-induced desensitization of neurons, indicating its role in protecting MOR against overstimulation. This alteration in the membrane lipid environment mediated by *PTCHD1* influences the trafficking dynamics of opioid receptors. Additionally, a cholesterol photoclick-reaction assay conducted on purified *PTCHD1* elution fractions by Hiltunen, and collaborators revealed a similar binding efficiency of the cholesterol to both glycosylated and native forms of *PTCHD1* (Hiltunen et al., 2023).

Recent investigations have also highlighted the interaction of *PTCHD1* with cholesterol sites and proteins from the cholesterol signaling pathway such as ATAD3A (Table 2; Arguello et al., 2021; Hiltunen et al., 2023). Interestingly, a study by Kinnebrew and collaborators suggest a mechanism by which *PTCH1* regulates SMO by altering the cholesterol distribution in cell membranes, offering insights into the cholesterol accessibility in membrane-dependent cellular processes (Kinnebrew et al., 2021). The *PTCHD* family member NPC1 also acts as an intracellular cholesterol transporter mediating the export of unesterified cholesterol (Fig. 1; Kunkel et al., 2023).

Functional studies on *PTCHD1* missense variants involved in NDDs, such as the p.(Pro32Arg) and p.(Gly303Arg), highlighted their detrimental effects on processing and stability. Specific structural evidence (similar positions to the residues in *Ptch1*, based on the *PTCH1* Cryo-EM assay) suggests a role in cholesterol transport like NPC1 and *PTCH1* (Xie et al., 2024). Importantly, although *PTCHD1* is frequently compared to the *PTCH1* receptor due to its conserved SSD motif and overall structural similarity, its role in cholesterol dynamics within neuronal compartments may be more closely aligned with that of NPC1. Both proteins localize to postsynaptic structures (Mitroi et al., 2019; Ung et al., 2018), participate in the trafficking and distribution of cholesterol (Maza et al., 2022; Hiltunen et al., 2023; Yoon et al., 2022), and have been implicated in neurodevelopmental disorders (Chaudhry et al., 2015; Tiscione et al., 2019), suggesting that NPC1 may provide a more relevant functional framework for understanding the role of *PTCHD1*.

These findings establish *PTCHD1* as a critical regulator of cholesterol homeostasis and membrane dynamics, with its Sterol Sensing Domain (SSD) enabling direct cholesterol binding and modulation of μ-opioid receptor trafficking. By influencing cholesterol content distribution, *PTCHD1* contributes to membrane-dependent cellular processes, including interactions with key cholesterol-associated proteins like ATAD3A and CAV1. These insights not only reinforce the pivotal role of *PTCHD1* in sterol regulation but also provide foundations to further explore its impact on neuronal physiology and disease mechanisms.

## 6.3. *PTCHD1* in the inflammation process through the Kynurenine pathway

*Ptch1* knockout mice exhibit significant changes in Kynurenine Pathway (KP) metabolite concentrations both in the serum and frontal cortex, suggesting the involvement of *PTCHD1* in the activated KP (Murakami et al., 2019). In fact, Kynurenine and its breakdown products play essential roles in blood vessel dilation during inflammation and immune response processes. These findings suggest a critical role for

*PTCHD1* in modulating the Kynurenine pathway, a key regulator of inflammation and immune responses. The altered metabolite concentrations in *Ptch1* knockout mice suggest that *PTCHD1* may influence neuroinflammatory processes and/or the vascular regulation, providing a novel link between the protein and the immune-metabolic axis.

# 7. Linking *PTCHD1* genetic variants to NDDs

## 7.1. *PTCHD1* in NDDs

In a recent comprehensive review, Pastore and collaborators have reported some of the neurodevelopmental contributions of the *PTCHD1* gene in ID and ASD (Pastore et al., 2022). In the following subsections, we highlight additional key findings regarding the genetic variations and clinical impacts of *PTCHD1* mutations in NDDs.

### 7.1.1. *PTCHD1* mutations in ID and ASD

*PTCHD1* genetic variants, including loss-of-function mutations and copy number variations (CNVs), have been strongly associated with NDDs, particularly ID and ASD. Clinical studies on *PTCHD1* non-syndromic mutations highlighted a spectrum of phenotypic manifestations (Fig. 4A). Based on the 23 patients with *PTCHD1* variations, Chaudry and collaborators reported a global developmental delay (78 %), ID (39 %) and ASD or ASD-like traits (43 %) (Chaudhry et al., 2015). Of note, a significant proportion of patients also presents with microcephaly or macrocephaly (31 %). Behavioral and neurological features such as hyper-aggressiveness (18 %), hypotonia (26 %), and impaired balance and gait abnormalities (22 %) are also frequently reported. Notably, nearly half of the affected individuals (48 %) exhibit secondary orofacial hypotonia and open-mouth posture, suggesting a recognizable clinical phenotype (Fig. 4A).

### 7.1.2. Spectrum of pathogenic variants and functional studies

Genomic investigations have identified a range of pathogenic *PTCHD1* variants, including missense mutations, frameshift variants, and CNVs affecting the Xp22.11 region (Noor et al., 2010; Chaudhry et al., 2015; Pinto et al., 2010; Torrico et al., 2015). Loss-of-function mutations in *PTCHD1* segregate with disease in an X-linked recessive pattern, underscoring its role in ASD and ID etiology. Despite extensive genetic screening, *PTCHD1* demonstrates intolerance to missense variations, with a significantly reduced number of observed missense variants compared to expectations (Z-score = 2). Functional studies suggest that specific *PTCHD1* missense mutations, particularly those affecting extracellular loops and transmembrane domains, lead to protein instability and impaired plasma membrane localization, ultimately disrupting the *PTCHD1* function (Table 3).

The ClinVar database (Updated 04/2025; https://www.ncbi.nlm.nih.gov/clinvar/) reports missense variants in *PTCHD1* within 140 VUS (variants of uncertain significance) and 4 likely pathogenic variants associated with NDDs including p.(Pro32Arg), p.(Lys181Thr), p. (Gly300Arg) and p.(Ala310Pro). They are comprising 31 variants impacting amino acids included in the first extracellular loop (amino acids 43–271), 36 missense variants are referenced in the second extracellular loop (amino acids 521–695) and 46 missense variants distributed along the 12 TM domains. Interestingly, 10 missense variants are present in an intracellular loop between TM 6–7 (amino acids 432–497).

Recent publications have provided empirical evidence for the pathogenicity of several *PTCHD1* missense variants by demonstrating their decreased protein stability and altered cellular localization (Halewa et al., 2021; Xie et al., 2024, Pastore et al., 2025). Using cycloheximide chase assays (*protein synthesis*; Xie et al., 2024) and immunostaining approaches for colocalization signals and thus subcellular localization (Halewa et al., 2021), they showed that the *PTCHD1* missense mutations p.(Pro32Arg), p.(Gly300Arg) and p.(Ala310Pro) trigger its degradation, while others, such as p.(Lys181Thr) and p.(Tyr213Cys), lead to *PTCHD1*
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Comparative overview of the consequences of *PTCHD1* depletion in human and mouse models.; A. Clinical phenotype observed in patients with *PTCHD1* mutations. B. Behavioral consequences of *Ptchd1* deletions in knockout mouse models. C, D. Cellular and functional consequences of C. Human-derived 2D or 3D models and, D. Mouse tissue or neurons. Blue: decreasing and red: increasing values.; Biorender was used for the schema design.</figcaption></figure>

retention in the endoplasmic reticulum, preventing its functional integration to the plasma membrane. Nevertheless the pathogenic mechanisms of the *PTCHD1* variants p.(Ser51Asn), p.(Leu73Phe), p. (Gln102Arg), p.(Val150Met), p.(Lys181Thr), p.(Thr602Ala), p. (Ile830Arg) remain unclear, despite their normal subcellular localization, which could suggest a disruptive cholesterol binding or defective interaction with (un)identified PTCHD1 partner.

Furthermore, additional missense mutations in *PTCHD1* have been reported more recently in lined with NDDs phenotype (See Table 3; Farley et al., 2024; Montanaro et al., 2024; Aspromonte et al., 2025). In fact, Farley and colleagues used an exciting approach based on human derived model to unravel a *PTCHD1* VUS (variant of uncertain significance) (PTCHD1 c.2489 T > G; p.(Ile830Arg), identified in a child with autistic behavior, developmental delay, and hypotonia (Farley et al., 2024). Using CRISPR gene editing and human iPSC-based neural

modeling, they detect subtle but significant alterations in synaptic transmission and muscle contraction pathways, providing new insights into PTCHD1's potential role in NDDs in human.

Altogether these findings highlight the importance and necessity of functional validation approaches, beyond *in silico* predictions, to classify *PTCHD1* variants of uncertain significance (VUS) and refine clinical diagnostic criteria.

## 7.2. Contribution of PTCHD1 to NDDs: insights from pathophysiological model studies

The absence of *PTCHD1* has been increasingly linked to a range of NDDs, with pathophysiological studies in animal and human-derived models shedding light on its critical role in brain function.
<table><caption>**Table 3**
Functional impact of *PTCHD1* missense variants linked with NDDs phenotype in patients based on literature analysis.</caption><thead><tr><th>Predicted affected region of the protein</th><th>PTCHD1 missense variants</th><th>Inheritance</th><th>Functional impact</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="5">TM domain 1</td><td>P32R</td><td>Mat</td><td>Reduced protein expression, defective membrane localization, ER retention<br>(Halewa et al., 2021; Pastore et al., 2022, 2025; Xie et al., 2024)<br>(Xie et al., 2024)</td><td></td></tr><tr><td>P32L</td><td>Mat</td><td>Reduced protein expression, defective membrane localization, ER retention<br>(Halewa et al., 2021)</td><td></td></tr><tr><td>S51N</td><td>NR</td><td><u>Not defined yet</u></td><td>(Torrico et al., 2015; Halewa et al., 2021; Pastore et al., 2025)<br>(Noor et al., 2010; Halewa et al., 2021)</td></tr><tr><td>L73F</td><td>Mat</td><td><u>Not defined yet</u></td><td>(Pastore et al., 2022; Xie et al., 2024)</td></tr><tr><td>P75Q</td><td>NR</td><td>Reduced protein expression, defective membrane localization, PTM defects, ER retention<br>(Pastore et al., 2025)</td><td></td></tr><tr><td>P75L</td><td>NR</td><td>Reduced protein expression, ER retention<br>(Pastore et al., 2025)</td><td></td></tr><tr><td>Q102R</td><td>NR</td><td><u>Not defined yet</u></td><td>(Pastore et al., 2025)</td></tr><tr><td>V150M</td><td>NR</td><td>Defective membrane localization<br><u>Defective membrane localization</u>, Synaptic localization<br>(Pastore et al., 2025)<br>(Noor et al., 2010; Halewa et al., 2021; Xie et al., 2024)</td><td></td></tr><tr><td>Extracellular loop 1</td><td>I173V</td><td>Mat</td><td>Similar protein expression, membrane localization, PTM defects<br>(Pastore et al., 2022; Xie et al., 2024)</td><td></td></tr><tr><td rowspan="5">TM domain 3</td><td>K181T</td><td>Mat</td><td>Reduced protein expression, defective membrane localization, ER retention<br>(Karaca et al., 2015; Halewa et al., 2021)</td><td></td></tr><tr><td>V195I</td><td>Mat</td><td>Reduced protein expression, defective membrane localization, ER retention, Synaptic localization<br>(Noor et al., 2010; Halewa et al., 2021; Pastore et al., 2025)</td><td></td></tr><tr><td>Y213C</td><td>Mat</td><td>Reduced protein expression, defective membrane localization, ER retention<br>(Halewa et al., 2021; Pastore et al., 2025)</td><td></td></tr><tr><td>G300R</td><td>Mat</td><td>Reduced protein expression, defective<br>(Halewa et al., 2021)</td><td></td></tr></tbody></table>

<table><caption>**Table 3 (continued)**</caption><thead><tr><th>Predicted affected region of the protein</th><th>PTCHD1 missense variants</th><th>Inheritance</th><th>Functional impact</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="5">TM domain 1</td><td>G303R</td><td>NR</td><td>membrane localization, ER retention<br>Reduced protein expression, defective membrane localization, PTM defects</td><td>(Xie et al., 2024)</td></tr><tr><td>A310P</td><td>De novo</td><td>Reduced protein expression, defective membrane localization, ER retention</td><td>(Halewa et al., 2021)</td></tr><tr><td>Intracellular loop 2</td><td>H359R</td><td>Mat</td><td><u>Not defined yet</u></td><td>(Noor et al., 2010; Halewa et al., 2021)</td></tr><tr><td>A470D</td><td>Mat</td><td><u>Not defined yet</u></td><td>(Noor et al., 2010; Halewa et al., 2021)</td></tr><tr><td>Intracellular loop 3</td><td>E479G</td><td>Mat</td><td><u>Not defined yet</u></td><td>(Noor et al., 2010; Halewa et al., 2021)</td></tr><tr><td rowspan="5">TM domain 3</td><td>F549C</td><td>NR</td><td>Membrane localization, ER retention<br>Reduced protein expression, defective membrane localization, PTM defects, ER retention</td><td>(Firth et al., 2009; Pastore et al., 2024)</td></tr><tr><td>T602A</td><td>NR</td><td>Defective membrane localization</td><td>(Pastore et al., 2022; Xie et al., 2024)</td></tr><tr><td>F626S</td><td>NR</td><td>N/A</td><td>(Pastore et al., 2025)</td></tr><tr><td>I830R</td><td>De novo</td><td>Change in gene signatures in synaptic transmission and muscle contraction pathways during differentiation (hiPSC to NPC)</td><td>(Montanaro et al., 2024)</td></tr></tbody></table>

### 7.2.1. Ptchd1 absence leads to cognitive alterations and hyperactivity phenotype in mice

Various knockout mouse models targeting different exons of the *Ptchd1* gene have been generated, revealing diverse behavioral phenotypes (Fig. 4B). *Ptchd1* knockout mouse models exhibit a variety of behavioral phenotypes, including hyperactivity, repetitive behaviors, cognitive deficits, decreased attention, impulsivity, increased aggressive behavior, and fragmented sleep (Fig. 4B). Interestingly, these mice show reduced attention, altered hippocampal-dependent spatial learning and memory, and deficits in fear conditioning paradigms (Murakami et al., 2019; Tora et al., 2017; Ung et al., 2018). For instance, latency in active place avoidance tasks, which depend on hippocampal-dependent spatial learning and memory, is reduced in full *Ptchd1* knockout adult male mice but remains normal in the *Sst-Cre* (Somatostatin-CRE) dependent
*Ptchd1* knockout model. This suggests that some phenotypes may be independent of somatostatin neurons, a subset of GABAergic neurons known to mark inhibitory interneurons (Wells et al., 2016; Song et al., 2021). Additionally, memory deficits observed in knockout mice specifically within the thalamic reticular nucleus (TRN) are not rescued by pharmacological treatment (Nakajima et al., 2019). However, specific knockdown of *Ptchd1* in the anterodorsal (AD) TRN region does not affect foot-shock-induced freezing during contextual fear conditioning (CFC) training but rather results in long-term memory recall deficits (Roy et al., 2021).

Moreover, *Ptchd1* knockout mice display significant deficits in contextual (hippocampal-dependent) fear conditioning paradigms (Wells et al., 2016; Ung et al., 2018). Although Wells and collaborators reported a deficit in cued amygdala-dependent fear conditioning (Wells et al., 2016) that was not observed in the more recent study (Ung et al., 2018). Notably, the *Ptchd1*⁻/⁻ mice show a significant reduction in cliff avoidance reaction, which may be indicative of ADHD-like behaviors. Furthermore, these knockout mice exhibit reduced anxiety-like behaviors and increased aggressiveness (Ung et al., 2018; Murakami et al., 2019; Wells et al., 2016).

These findings from *Ptchd1* knockout mouse models underscore the critical role of this gene in regulating neural circuits involved in socio-cognitive and emotional behaviors. These models exhibit a range of behavioral and cognitive alterations that parallel symptoms of NDDs, offering a platform for studying potential therapeutic interventions. However, further research is still needed to elucidate the underlying mechanisms and explore potential therapeutic interventions targeting the pathways impacted by *Ptchd1* mutations.

### 7.2.2. *Derived human neuronal models of PTCHD1 alteration highlight the synaptic function of PTCHD1*

Advances in stem cell and organoid technologies have transformed our understanding of the functional consequences of gene deficiencies associated with NDDs. By leveraging patient-derived induced pluripotent stem cells (iPSCs) and organoids, researchers can explore the cellular and molecular impacts of specific gene deletions in details.

Ross and collaborators investigated the functional consequences of *PTCHD1* and *PTCHD1-AS* (*Anti-sense*) locus deletions using iPSCs derived from both unaffected control subjects and ASD patients (Fig. 4C; Ross et al., 2020). Through molecular and electrophysiological approaches, they evaluated the function of iPSC-derived cortical neurons and exhibited reduced miniature excitatory postsynaptic current frequency and *N*-methyl-D-aspartate receptor. Additionally, Kiral and collaborators utilized ASD patient-derived iPSCs lacking the antisense non-coding RNA *PTCHD1-AS* to generate 2D neuronal cultures. Despite the absence of *PTCHD1-AS* affecting *PTCHD1* transcription, these neurons exhibited reduced miniature excitatory postsynaptic current frequency (Farley et al., 2024), suggesting a potential role in ASD pathology, a finding also highlighted in the *Ptchd1* knockout mouse model by Ung and collaborators. Beyond iPSCs, researchers have begun exploring the use of human embryonic stem cells (hESCs) to generate ventral thalamic organoids (vThOs) with a thalamic reticular nucleus (TRN) signature (Kiral et al., 2023). Perturbations in *PTCHD1* were found to impair neuronal functions within the vThOs, although they did not significantly affect the overall thalamic lineage development. This study highlights the specific roles of *PTCHD1* within thalamic organoid models, providing insights into its potential contributions to NDDs.

The human-derived models, including iPSCs and organoids, have illuminated the pivotal role of *PTCHD1* in neuronal and synaptic transmission, offering critical insights into its contribution to NDDs. Studies using patient-derived iPSCs and organoids also revealed how *PTCHD1* dysfunction impacts synaptic activity, excitatory neurotransmission, and thalamic neuronal function, bridging molecular alterations to functional outcomes. These models not only highlight the specific role of *PTCHD1* in neuronal and synaptic pathways but also underscore the broader utility of stem cell and organoid technologies in unraveling the

complexities of gene-specific contributions to NDDs. This progress lays a promising foundation for developing targeted, personalized therapeutic strategies to address *PTCHD1*-related pathologies.

## 8. Conclusion

NDD encompass a wide array of conditions marked by deficits in cognitive, social, and motor functions arising from disrupted brain development, with ID, ASD and ADHD being prominent examples. Recent genetic studies have identified over 1500 candidate genes associated with these disorders, revealing common pathophysiological pathways involving synaptic signalling, chromatin remodelling, and cytoskeleton signalling. Among these genes, *PTCHD1*, located on the X chromosome, has emerged as a key player, with mutations and deletions linked to non-syndromic NDDs characterized by global developmental delays, ID, ASD features, and various behavioural issues.

Investigations into *PTCHD1* have unveiled its expression in critical brain regions, particularly during early neurogenesis and synaptogenesis stages, with a distinct peak in the Thalamic Reticular Nucleus (TRN) observed during early postnatal development. Despite initial speculations regarding the involvement of *PTCHD1* in the Sonic Hedgehog (SHH) pathway, recent findings refute its role in SHH canonical signalling, instead highlighting its affinity for cholesterol binding and its impact on membrane cholesterol content, suggesting a potential non-canonical pathway. Additionally, the association of *PTCHD1* with synaptic scaffolding proteins or the retromer complex underscores its multifaceted roles at the synapse, implicating the protein in the regulation of the synaptic structure, function, and plasticity. The emerging evidence that *PTCHD1* interacts with ribonucleoproteins and stress granule proteins, as well as its novel roles in cholesterol regulation, addiction, and inflammation leave the possibility that mutations in the *PTCHD1* gene may lead to additional deleterious pathologies.

In this review, we have underscored the critical role of *PTCHD1* at the synapse. However, its precise molecular function remains poorly understood. As a perspective, it will be crucial to address several key questions moving forward. Even though the transmembrane protein *PTCHD1* is sharing structural homology with the PATCHED receptor, it has been well established that they act differently on canonical Hedgehog pathway. Further evidence also suggests a close association between *PTCHD1* and NPC1, another actor of the PATCHED family, likely due to their shared roles in cholesterol metabolism. Although this should be rigorously evaluated and has the potential to lead to an exciting and groundbreaking line of research.

Yet, *PTCHD1* remains an orphan receptor, with the identification of its ligand posing a critical yet formidable challenge. Along the same lines, it will be worth carefully exploring their subcellular localization at synapses, similarly to NPC1, which is known to reside in lysosome-related organelles, in order to clarify the potential interplay between both proteins. Unraveling this interaction is essential for advancing targeted therapeutics and the development of specific agonists. Additionally, further research should aim to elucidate the downstream signaling pathways regulated by *PTCHD1*, which could provide deeper mechanistic insights into its role in synaptic function and define molecular and functional indicators for clinical transfer and towards potential therapeutic applications.

## Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT in order to refine the scientific English. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.
# Declaration of Competing Interest

The authors declare no competing interests.

# Acknowledgements

This work was supported by the 'Agence Nationale de la Recherche' (ANR-20-CE17-0006) and by the 'Association pour le développement de la neurogénétique'.

D.C. Ung. wrote the original draft of the review manuscript. All authors edited and commented on the manuscript.

# Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.neubiorev.2025.106307.

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## Glossary

**ADHD:** Attention Deficit Hyperactivity Disorder

**AMPAR:** α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor

**AS:** Antisense

**ASD:** Autism Spectrum Disorder

**CA1:** Cornu Ammonis 1 (subregion of the hippocampus)

**CNV:** Copy Number Variant

**CX:** Cortex

**DG:** Dentate Gyrus (region of the hippocampus)

**DISP1:** Dispatched Homolog 1

**GST:** Glutathione S-Transferase

**hESC:** Human Embryonic Stem Cells

**ID:** Intellectual Disability

**IPSC:** Induced Pluripotent Stem Cells

**KO:** Knockout

**MAGUK:** Membrane-Associated Guanylate Kinase

**mGluR:** Metabotropic Glutamate Receptor

**NDDs:** Neurodevelopmental Disorders

**NMDAR:** N-Methyl-D-Aspartate Receptor

**NPC1:** Niemann-Pick Disease, Type C1

**NPCs:** Neural Progenitor Cells

**PDZ:** PSD95, Dlg, ZO-1 domain (a protein interaction module found in signaling proteins)

**Post:** Postsynaptic part

**Pre:** Presynaptic part

**PSD:** Postsynaptic Density

**PSD95:** Postsynaptic Density Protein 95

**PTCH:** Patched

**PTCHD1:** Patched Domain Containing 1

**PTM:** Post-Translational Modifications

**RNA:** Ribonucleic Acid

**SAP102:** Synapse-Associated Protein 102

**SHH:** Sonic Hedgehog

**SMO:** Smoothened

**TGN:** Trans-Golgi Network

**TRN:** Thalamic Reticular Nucleus

**VUS:** Variant of Uncertain Significance

**vThOs:** Ventral Thalamic Organoids