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1-Deoxysphingolipids (1-deoxySLs)

Summary

1-Deoxysphingolipids (1-deoxySLs) are atypical, bioactive sphingolipid species synthesized when serine palmitoyltransferase (SPT) mis-incorporates L-alanine instead of L-serine [1]. Lacking the canonical C1-hydroxyl group, 1-deoxySLs cannot undergo phosphorylation to sphingosine-1-phosphate (S1P) for degradation by S1P lyase, nor can they form complex glycosphingolipids [1]. A genome-wide CRISPRi screen combined with isotopic tracer lipidomics revealed that 1-deoxySL toxicity is not intrinsic to the free long-chain base, but requires elongation into very long-chain (VLC) 1-deoxydihydroceramides—specifically nervonyl-1-deoxyDHCer (m18:0/24:1) and lignoceryl-1-deoxyDHCer (m18:0/24:0)—by ELOVL1 and CERS2, which trigger mitochondrial membrane permeabilization, mPTP opening, BAX activation, and axonal degeneration [1].

Enzymatic Synthesis & Bifurcated Metabolic Fate

                                Palmitoyl-CoA + L-Alanine
                                           │
                                           │ SPTLC1 / SPTLC2 (SPT)
                                           │ [Mutations: C133W, C133Y, V144D; A182P]
                                           ▼
                                 1-Deoxysphinganine
                                 (1-deoxySa, m18:0)
                                           │
                    ┌──────────────────────┴──────────────────────┐
                    │                                             │
                    │ CERS5 / CERS6 (LC)                          │ CERS2 + ELOVL1 (VLC)
                    ▼                                             ▼
        LC-1-deoxyDHCer (m18:0/16:0)                  VLC-1-deoxyDHCer (m18:0/24:0, 24:1)
                    │                                             │
                    │ FADS3 desaturation                          │ Accumulation in mitochondrial
                    │ Δ14(Z) double bond                          │ outer membrane
                    ▼                                             ▼
         Detoxified 1-deoxySo                         Mitochondrial respiration collapse,
         (Non-Toxic: IC50 > 3 µM)                     mPTP opening, BAX activation,
                                                      Sensory Axon Degeneration! [1]
  1. Condensation: Missense mutations in SPTLC1 (e.g. C133W, C133Y, V144D) or SPTLC2 (e.g. A182P) alter the substrate specificity of SPT, causing it to condense palmitoyl-CoA with L-alanine to form 1-deoxysphinganine (1-deoxySa, m18:0) [1]. In diabetes, low circulating L-serine to L-alanine ratios drive similar ectopic 1-deoxySa synthesis [1].
  2. N-Acylation & Elongation: 1-deoxySa is N-acylated by ceramide synthases (CERS) to form 1-deoxydihydroceramide (1-deoxyDHCer) species [1]. The fatty acid acyl chain length is governed by ELOVL1 (which elongates $C_{20}$ to $C_{22}, C_{24}$, and $C_{26}$ acyl-CoAs) and CERS2 [1].
  3. Isotopic Flux Lipidomics ($d_3$-1-deoxySa): Tracing $d_3$-1-deoxySa in HeLa cells treated with the CERS inhibitor Fumonisin B1 (FB1) resulted in massive accumulation of unacylated $d_3$-1-deoxySa and $d_3$-1-deoxySo long-chain bases, while completely suppressing downstream $d_3$-1-deoxyDHCer and $d_3$-1-deoxyCer [1]. FB1 treatment conferred complete protection against cell death, proving that the unacylated LCB is benign and N-acylation is required for cytotoxicity [1].
  4. Alternative Desaturation by FADS3: Canonically, dihydroceramides are desaturated at $\Delta 4E$ by DEGS1 to form ceramides [1]. However, 1-deoxyDHCer is not a substrate for DEGS1 [1]. Instead, fatty acid desaturase 3 (FADS3) introduces a $\Delta 14Z$ double bond into the sphingoid base to produce 1-deoxysphingosine (1-deoxySo, m18:1 $\Delta 14Z$) [1].
  5. Desaturation-Driven Detoxification: Supplementation with 1-deoxySo (m18:1) exhibits no cytotoxicity ($IC_{50} > 3.0\,\mu M$), whereas 1-deoxySa (m18:0) is highly toxic ($IC_{50} = 0.33\text{--}0.39\,\mu M$) [1]. FADS3-mediated $\Delta 14Z$ desaturation functions as an essential endogenous detoxification system [1].

Genome-Wide CRISPRi Screen Identifies Fatty Acid Elongation Pathway

Majcher et al. (2025) conducted a genome-wide CRISPR interference (CRISPRi) survival screen in K562 cells harboring dCas9-KRAB challenged with an $IC_{50}$ dose of 1-deoxySa ($1.5\,\mu M$) to map genetic modulators of 1-deoxySL toxicity [1]:

Gene Symbol Gene Name Functional Class / Pathway Phenotype under 1-deoxySa Challenge
ELOVL1 Elongation of very long chain fatty acids protein 1 Fatty acid elongation ($C_{20} \to C_{24}$) Top Protective Hit ($Z > +6.0$); knockout confers resistance [1].
CERS2 Ceramide synthase 2 VLC-specific sphingoid N-acylation Top Protective Hit; knockout confers total resistance [1].
ACACA Acetyl-CoA carboxylase alpha De novo fatty acid synthesis rate-limiting step Protective hit; reduces malonyl-CoA supply for elongation [1].
HSD17B12 17-Beta-hydroxysteroid dehydrogenase 12 3-Ketoacyl-CoA reductase step of FA elongation Protective hit [1].
PTPLB / HACD7 3-Hydroxyacyl-CoA dehydratase 7 Dehydratase step of FA elongation Protective hit [1].
FADS3 Fatty acid desaturase 3 $\Delta 14Z$ base desaturation Top Sensitizing Hit ($Z < -5.0$); silencing increases toxicity [1].

Isoform Specificity & Ceramide Synthase Dissection

While knockout of CERS2 (which selectively utilizes $C_{22:0}, C_{24:0}$, and $C_{24:1}$ acyl-CoAs) shifted the 1-deoxySa $IC_{50}$ from $0.33\,\mu M$ to $1.76\text{--}2.06\,\mu M$ (providing a 5- to 6-fold survival rescue), knockout of CERS5 or CERS6 (which synthesize long-chain species like $C_{16:0}$ and $C_{18:0}$) provided zero protection [1]. This demonstrates that toxicity is mediated exclusively by very long-chain (VLC) N-acyl chains [1].

Small-Molecule ELOVL1 Inhibition (Compound 22) Rescues Mitochondrial & Neuronal Function

To test pharmacological translation, Majcher et al. evaluated Compound 22 (CAS 2761063-99-2), a pyrimidine-ether small-molecule inhibitor of ELOVL1 [1].

                     ┌─────────────────────────────────────────┐
                     │     1-deoxySa Challenge (0.5 µM)        │
                     │  - OCR (Respiration):     Marked Drop   │
                     │  - ECAR (Glycolysis):     Marked Drop   │
                     │  - mPTP Opening:          Triggered     │
                     └────────────────────┬────────────────────┘
                                          │
                                          │ Co-treatment with Compound 22 (1 µM)
                                          ▼
                     ┌─────────────────────────────────────────┐
                     │   Seahorse XF Mito Tox Assay Rescue     │
                     │  - Full OCR & ECAR Restoration          │
                     │  - VLC 1-deoxyDHCer Shift to LC         │
                     │  - Chicken DRG Sensory Axons Saved!     │
                     └─────────────────────────────────────────┘

Seahorse XF Mito Tox Assay & Mitochondrial Rescue

  • Mitochondrial Respiration: Treatment of HeLa cells with 1-deoxySa ($0.5\,\mu M$) caused a catastrophic loss of oxygen consumption rate (OCR), maximal respiratory capacity, and glycolytic capacity (ECAR) [1].
  • Seahorse Restoration: Co-treatment with Compound 22 ($1.0\,\mu M$) completely restored all mitochondrial respiratory parameters and ECAR to vehicle baseline levels [1].
  • Mitochondrial Pore Mechanism: VLC 1-deoxyDHCer integrates into the outer mitochondrial membrane, inducing membrane permeabilization, BAX translocation, and opening of the mitochondrial permeability transition pore (mPTP) [1]. Compound 22 shifts lipid profiles from VLC ($m18:0/24:1$) to LC ($m18:0/16:0$), which are benign to mitochondrial membranes [1].

Primary Neuronal Protection in Chicken Embryo DRGs

In primary dorsal root ganglion (DRG) sensory neurons isolated from day-9 chicken embryos, 1-deoxySa administration induced severe axonal fragmentation and swelling [1]. Compound 22 suppressed VLC 1-deoxyDHCer synthesis and rescued sensory axonal integrity [1].

Significance

This work reframes 1-deoxySL neuropathies (including HSAN1 and diabetic peripheral neuropathy): toxicity is not driven by the initial synthesis of 1-deoxysphinganine, but by its downstream metabolic conversion into very long-chain (VLC) species by ELOVL1 and CERS2 [1]. Inhibiting ELOVL1 represents a precision therapeutic target that neutralizes neurotoxicity while preserving canonical sphingolipid pools [1].

  • Hornemann Lab — investigates atypical sphingolipid synthesis, structure, and neurotoxicity mechanisms.

Citations

[1] Majcher, A., Karsai, G., Yusifov, E., Schaettin, M., Malagola, E., Horvath, P., Li, J., Rodriguez-Gallardo, S., Shimizu, K., Zhibo, G., Dubey, R., Peterson, T., Harayama, T., & Hornemann, T. (2025). Very long-chain fatty acids drive 1-deoxySphingolipid toxicity. Nature Communications, 16, 11650. DOI: 10.1038/s41467-025-66687-8. Source: 41467_2025_Article_66687.pdf. Supports: Genome-wide CRISPRi screen parameters (K562, 1.5 uM 1-deoxySa), d3-isotope flux lipidomics (FB1 LCB trapping), top resistance genes (ELOVL1, CERS2, ACACA), top sensitizing gene (FADS3), 1-deoxySo IC50 > 3 uM, Compound 22 (CAS 2761063-99-2) Seahorse Mito Tox rescue, and chicken embryo DRG neuronal axon protection. Location: Full text -- Abstract, Results, Figures 1-5. Verified 2026-07-30.