RAB23 Human

What is RAB23 and what is its functional significance in human cells?

RAB23 is a small GTPase belonging to the RAS oncogene family that plays crucial roles in membrane trafficking and protein transport in eukaryotic cells. It functions as a negative regulator of the Sonic Hedgehog (Shh) signaling pathway, which is essential for embryonic development . Beyond development, RAB23 has emerged as a significant factor in cellular processes including ciliary trafficking, left-right patterning, and potentially in autophagy mechanisms . The gene is located on chromosome 6p12.1-q12 in the human genome, and its expression is regulated by various factors including miRNAs that control its expression epigenetically .

What is Carpenter Syndrome and how is it related to RAB23?

Carpenter Syndrome (CS) is a rare autosomal recessive disorder characterized by craniosynostosis (premature fusion of cranial sutures), polysyndactyly (extra digits and digit fusion), skeletal defects, obesity, and intellectual disability . CS results from loss-of-function mutations in the RAB23 gene. The incorrect embryogenesis underlying CS is believed to occur between days 30 and 49 of embryonic development . Although RAB23 defects cause CS in an autosomal recessive inheritance pattern, there is marked intrafamilial variability in phenotypic expression .

What cellular processes are regulated by RAB23?

RAB23 regulates multiple cellular processes including:

• Membrane trafficking and vesicular transport

• Ciliary structure formation and function

• Hedgehog signaling pathway regulation

• Nodal signaling in left-right patterning

• Potential roles in autophagy processes, particularly in antibacterial defense

• Embryonic development, especially in cranial suture formation and limb development

These various functions explain the diverse phenotypic impacts of RAB23 mutations in diseases like Carpenter Syndrome.

How does RAB23 regulate the Sonic Hedgehog signaling pathway?

RAB23 functions as a negative regulator of the Sonic Hedgehog (Shh) signaling pathway, which is critical for embryonic development and patterning . This regulation occurs at the level of the primary cilium, where RAB23 influences the trafficking of Shh pathway components . In normal cellular function, RAB23 helps maintain appropriate levels of Shh signaling by ensuring proper ciliary localization of pathway components and potentially regulating their activity. When RAB23 is absent or dysfunctional, as in Carpenter Syndrome, this regulatory control is lost, leading to altered Shh signaling dynamics and subsequent developmental abnormalities . Recent research using RAB23-knockout neural progenitor cells demonstrated that these cells were desensitized to primary cilium-dependent activation of the Hedgehog signaling pathway .

What is the role of RAB23 in left-right patterning during embryonic development?

RAB23 plays a crucial role in establishing left-right asymmetry during embryonic development through the Nodal signaling pathway. While RAB23 is not required for initial symmetry breaking in the node, it is essential for the expression of Nodal and Nodal target genes in the left lateral plate mesoderm (LPM) . Research using experimental approaches like microinjection of Nodal protein and transfection of Nodal cDNA has shown that RAB23 is specifically required for the production of functional Nodal signals rather than the cellular response to these signals . This function appears to be conserved across species, as similar roles have been demonstrated in zebrafish, where RAB23 is required in Kupffer's vesicle (the teleost equivalent of the mouse node) . Importantly, this role in left-right patterning is independent of the Shh pathway, highlighting RAB23's diverse functions in development .

How does RAB23 contribute to ciliary function and structure?

RAB23 contributes to ciliary function and structure in a cell-type dependent manner . Recent research using conditional knockout mouse models, patient-derived induced pluripotent stem cells (iPSCs), and zebrafish morphants has demonstrated that RAB23 loss leads to context-dependent ciliary abnormalities . In RAB23-deficient models:

• Neocortical neurons show significant reduction in ciliation frequency

• Chondrocytes exhibit altered cilia length and volume

• Neural progenitor cells derived from CS patient iPSCs show reduced ciliation frequency and/or shortened primary cilia

• Some cell types (epithelial cells, cerebellar granule cells, and hippocampal neurons) maintain relatively normal ciliation

This cell-type specific impact explains the selective manifestation of symptoms in Carpenter Syndrome patients, where certain tissues and developmental processes are affected while others remain relatively normal .

What specific mutations in RAB23 have been documented in Carpenter Syndrome patients?

At least 12 different mutations in the RAB23 gene have been documented in Carpenter Syndrome patients . These include:

• A mutation that eliminates the acceptor splice site of exon 5, resulting in deletion of 8 nucleotides in the Rab23 mRNA, causing a frameshift and premature termination codon at position 161 (p.V161fsX3)

• A homozygous mutation (c.481G>C) leading to skipping of exon 6 and premature termination codon (p.Val161Leufs16)

• A duplication mutation (c.86dupA) found in the homozygous state in four relatives of Comorian origin

These mutations typically result in loss of function of the RAB23 protein through frameshift and premature termination, preventing proper protein production and function . The majority of documented mutations lead to complete loss of functional RAB23 protein, explaining the recessive inheritance pattern of the syndrome.

How variable is the clinical presentation of Carpenter Syndrome, and what explains this variability?

Carpenter Syndrome shows marked intrafamilial variability in clinical presentation, even among patients with identical RAB23 mutations . Key aspects of this variability include:

• Severity of craniosynostosis ranging from cloverleaf skull to primarily involving metopic ridge

• Variable presentations of polydactyly and syndactyly in hands and feet

• Differences in mental development (generally normal, though some patients show impaired motor development)

• Variable presence of additional features like hydrocephalus

The variability likely stems from the context-dependent functions of RAB23, as demonstrated in recent research showing cell-type specific effects of RAB23 deficiency on ciliary function . Other genetic and environmental factors may also influence phenotypic expression, creating a spectrum of clinical manifestations even within the same family carrying identical RAB23 mutations.

What experimental models are currently available for studying RAB23 function?

Several experimental models have been developed to study RAB23 function, particularly in the context of Carpenter Syndrome:

• Conditional knockout (CKO) mouse models: These include global gene knockout mutants (using actin-Cre) and neural progenitor cell-specific knockout mutants, which display developmental and phenotypic abnormalities resembling clinical features of CS and ciliopathies .

• Patient-derived induced pluripotent stem cells (iPSCs): Reprogrammed from biopsies of CS patients, these cells can be differentiated into various cell types to study cell-type specific effects of RAB23 mutations .

• Zebrafish morphants: RAB23-silenced zebrafish (rab23 MO) show perturbation of ciliation primarily in the rostral neural tube but not in other regions like the caudal spinal cord neurons .

• Cell culture models: Various cell lines with RAB23 overexpression or knockdown have been used to study specific functions, such as the role of RAB23 in UVB-induced autophagy in HaCaT cells .

These diverse models allow researchers to investigate RAB23 function at multiple levels, from molecular mechanisms to developmental processes and disease pathogenesis.

What methodologies are recommended for investigating RAB23's potential role in autophagy?

To investigate RAB23's role in autophagy, researchers should consider a multi-faceted approach:

• Molecular markers assessment: Measure autophagy flux markers like LC3-II and Beclin1 using immunoblotting in RAB23 overexpression and knockdown models. Studies have shown that RAB23 overexpression increases LC3-II and Beclin1 expression after UVB exposure, while RAB23 knockdown decreases their expression .

• Fluorescence microscopy: Quantify autophagosome formation using fluorescent markers for autophagosomes. Previous research demonstrated that RAB23 overexpression significantly increases autophagosome numbers .

• Infection models: Use bacterial infection models to assess RAB23's role in antibacterial autophagy. Studies with Group A Streptococcus (GAS) have shown that RAB23 is required for GcAV (GAS-containing autophagosome-like vacuoles) formation .

• Signaling pathway analysis: Investigate RAB23's connection to known autophagy signaling pathways, as this relationship remains poorly understood .

• Stress induction: Apply various autophagy-inducing stressors (starvation, UV exposure, bacterial infection) to determine context-specific roles of RAB23 in different autophagy scenarios .

It's important to note that RAB23 appears to function differently in stress-induced autophagy versus starvation-induced autophagy, highlighting the need for multiple experimental conditions .

How can researchers effectively use patient-derived iPSCs to study RAB23 function?

Patient-derived induced pluripotent stem cells (iPSCs) offer powerful tools for studying RAB23 function in human cellular contexts. Effective methodology includes:

• Reprogramming and validation: Obtain patient biopsies with confirmed RAB23 mutations and reprogram them into iPSCs using established protocols. Validate pluripotency markers and confirm the RAB23 mutation is preserved .

• Directed differentiation: Differentiate iPSCs into relevant cell types affected in Carpenter Syndrome, particularly neural progenitor cells, neurons, chondrocytes, and other lineages of interest .

• Comparative analysis: Always include control iPSCs from healthy donors differentiated in parallel to properly assess phenotypic differences.

• Ciliary analysis: Examine primary cilia using immunofluorescence microscopy with markers such as acetylated α-tubulin (ciliary axoneme) and γ-tubulin (basal body), assessing parameters like ciliation frequency, cilia length, and morphology .

• Functional assays: Test Hedgehog pathway responsiveness using agonists like SAG or SHH ligand to evaluate if differentiated cells from CS patients show altered pathway activation .

• Rescue experiments: Perform genetic rescue by reintroducing wild-type RAB23 to confirm phenotypes are specifically due to RAB23 deficiency.

• Single-cell analyses: Consider single-cell transcriptomics to capture cell-type specific effects of RAB23 deficiency, given its context-dependent functions .

This approach has already yielded important insights, demonstrating reduced ciliation frequency and/or shortened primary cilia in neurons and neural progenitor cells derived from CS patient iPSCs .

How do researchers distinguish between Shh-dependent and Shh-independent functions of RAB23?

Distinguishing between Sonic Hedgehog (Shh)-dependent and Shh-independent functions of RAB23 requires sophisticated experimental approaches:

• Genetic interaction studies: Using double knockout/knockdown models of RAB23 and components of the Shh pathway. If phenotypes are rescued or exacerbated compared to single mutants, this suggests interaction between pathways.

• Pathway-specific readouts: Measuring activation of Shh pathway targets (like Gli transcription factors) versus non-Shh pathway targets (like Nodal pathway components) in RAB23 mutant backgrounds .

• Tissue-specific analyses: Examining RAB23 functions in tissues where Shh signaling is minimal or absent. For example, RAB23's role in left-right patterning via the Nodal pathway has been shown to be independent of Shh signaling .

• Biochemical approaches: Using co-immunoprecipitation and proximity ligation assays to identify RAB23 interacting partners that are either part of or independent from the Shh pathway.

• Rescue experiments: Testing whether Shh pathway modulation can rescue specific phenotypes in RAB23-deficient models. Phenotypes that cannot be rescued by Shh pathway modulation likely represent Shh-independent functions.

Research has already established that RAB23's role in transmitting asymmetric patterning information from the node to the lateral plate mesoderm is independent of the Shh pathway , demonstrating the importance of this distinction.

What are the cell-type specific effects of RAB23 deficiency on ciliary structure and function?

RAB23 deficiency impacts ciliary structure and function in a strikingly cell-type specific manner, as demonstrated by recent research using multiple model systems :

• Neocortical neurons: Exhibit significantly reduced ciliation frequency in RAB23 conditional knockout mice and patient-derived iPSC models .

• Chondrocytes: Show altered cilia length and volume but maintain relatively normal ciliation frequency .

• Neural progenitor cells: Derived from CS patient iPSCs display both reduced ciliation frequency and shortened primary cilia .

• Epithelial cells: Maintain relatively normal ciliation despite RAB23 deficiency .

• Cerebellar granule cells and hippocampal neurons: Show minimal changes in ciliation frequency .

• Zebrafish neural cells: Display regional differences, with ciliation primarily disrupted in the rostral neural tube but preserved in caudal spinal cord neurons .

This cell-type specificity likely explains the selective nature of developmental abnormalities observed in Carpenter Syndrome, where certain tissues and structures are affected while others develop normally despite the systemic mutation . Understanding the molecular basis of this context-dependent function represents an important frontier in RAB23 research.

What are the future research priorities for understanding RAB23 function in human disease?

Based on current knowledge gaps, several research priorities emerge for advancing understanding of RAB23 function in human disease:

• Mechanistic understanding of ciliary regulation: Further investigation is needed to understand how RAB23 controls ciliary trafficking in a cell-type specific manner. Key questions include how RAB23 nucleotide state is controlled and how it directs receptor delivery to the ciliary membrane .

• Conditional animal models: Development of additional conditional transgenic or knockout animals would enable more precise spatial and temporal control for analyzing RAB23 expression and function in specific tissues and developmental stages .

• Autophagy pathway connections: More convincing evidence is needed to directly prove RAB23's role in autophagy pathways and to understand how it connects to established autophagy signaling mechanisms .

• Therapeutic approaches: Exploring potential therapeutic interventions for Carpenter Syndrome based on pathway modulation, particularly investigating whether targeting downstream effectors of RAB23 could mitigate developmental defects.

• Broader physiological roles: Investigating what broader physiological roles the discrete RAB23-dependent ciliary targeting mechanism has beyond development could reveal new disease associations .

• Additional mutation identification: Further identification of RAB23 mutations in patients with CS-like phenotypes could expand understanding of genotype-phenotype correlations .

Addressing these research priorities will not only advance understanding of RAB23 biology but could also provide insights into fundamental mechanisms of ciliary function, development, and disease pathogenesis.