Complex Hereditary Spastic Paraplegia Cell Models for Research

Hereditary Spastic Paraplegia (HSP) is a group of inherited neurodegenerative disorders characterized by progressive spasticity and weakness of the lower limbs. Complex HSP refers to forms with additional neurological or systemic features. The global prevalence of HSP is estimated at 1-10 per 100,000 individuals (WHO, 2023). Complex forms account for approximately 20-30% of all HSP cases. The disease typically manifests in adulthood, but childhood onset occurs in some subtypes. There is no cure, and current management is symptomatic. The clinical impact is significant, with many patients requiring mobility aids and experiencing reduced quality of life. Research into the molecular mechanisms is crucial for developing targeted therapies.

Complex HSP is an ideal model for studying axonal degeneration, intracellular trafficking, and mitochondrial dysfunction. The genetic heterogeneity (over 80 loci identified) provides a rich landscape for functional genomics. Public datasets such as the HSP Registry and the International HSP Genetics Consortium offer extensive clinical and genetic data. Open questions include the precise pathomechanisms linking mutations to neuronal dysfunction and the development of biomarkers for disease progression. Gene-edited cell models enable controlled experiments to dissect these pathways.

• • Although HSP is not a cancer, the term 'carcinogenic' is not applicable. Instead, we focus on key pathogenic pathways:
• • Axonal transport defects: Mutations in motor proteins (e.g., KIF5A) and adaptors (e.g., SPG11) impair anterograde and retrograde transport.
• • Endoplasmic reticulum (ER) shaping: Proteins like SPAST (spastin) and ATL1 (atlastin) are involved in ER morphogenesis; mutations disrupt ER network and lead to axonal degeneration.
• • Mitochondrial dysfunction: Altered mitochondrial dynamics and bioenergetics are observed in several HSP subtypes.
• • Lipid metabolism: Genes like FA2H and CYP7B1 affect lipid synthesis and degradation, impacting myelin integrity.
• • Autophagy and lysosomal function: SPG11 and SPG15 are involved in autophagic clearance; defects lead to accumulation of toxic aggregates.

| Gene | Frequency (%) | Mutation Type | Functional Effect |

|------|---------------|---------------|-------------------|

| SPAST (SPG4) | 40% | Missense, truncating | Impaired microtubule severing, ER shaping |

| SPG11 | 10% | Frameshift, nonsense | Defective autophagy, axonal transport |

| ATL1 (SPG3A) | 10% | Missense | Disrupted ER fusion, axonal trafficking |

| KIF5A (SPG10) | 5% | Missense | Impaired kinesin motor function |

| REEP1 (SPG31) | 5% | Missense, truncating | ER shaping defects |

| ZFYVE26 (SPG15) | 5% | Frameshift | Impaired autophagy, endosomal trafficking |

Data from TCGA (not applicable) and COSMIC (not applicable) – these are germline mutations from ClinVar and literature.

• • Key signaling networks affected in complex HSP:
• • MAPK/ERK pathway: Altered in response to cellular stress, contributing to neurodegeneration.
• • PI3K/AKT/mTOR pathway: Dysregulated in some subtypes, affecting autophagy and cell survival.
• • Wnt signaling: Involved in axonal guidance and synaptic function; mutations in HSP genes may disrupt this pathway.
• • Calcium signaling: Mitochondrial dysfunction leads to calcium overload, triggering apoptosis.
• • Key nodes: SPAST, SPG11, ATL1, KIF5A, REEP1, ZFYVE26.

| Cell Line | Origin | Key Mutations |

|-----------|--------|---------------|

| SH-SY5Y | Human neuroblastoma | Wild-type; can be edited |

| SK-N-SH | Human neuroblastoma | Wild-type; can be edited |

| NSC-34 | Mouse motor neuron-like | Wild-type; can be edited |

| iPSC-derived neurons | Patient-derived | Patient-specific mutations |

| 3D organoids | iPSC-derived | Patient-specific mutations |

Organoids offer a more physiologically relevant 3D architecture and cell-cell interactions, enabling study of axonal degeneration and network activity.

• • PDX (Patient-Derived Xenograft): Not commonly used for HSP as it is not a cancer; but for research, patient-derived iPSC can be transplanted into mice.
• • GEMM (Genetically Engineered Mouse Models): Knock-in mice carrying patient mutations (e.g., SPAST, SPG11) recapitulate disease phenotypes.
• • Induced models: Use of viral vectors to express mutant genes in wild-type mice.
• • Zebrafish models: Used for high-throughput drug screening.
• • Drosophila models: Useful for genetic interaction studies.

• • CRISPR-Cas9 gene editing enables the creation of isogenic cell lines with precise mutations in HSP genes. For example:
• • SPAST knockout SH-SY5Y cells: Model loss-of-function mutations, useful for studying ER shaping defects.
• • SPG11 knockout iPSC-derived neurons: Model autophagy dysfunction.
• • KIF5A point-mutation knock-in cells: Model dominant-negative effects.
• • These models are commercially available from various sources, sequence-verified, and can be used for drug screening and mechanistic studies. They offer a controlled genetic background, reducing variability.

• • Gene-edited cell lines are essential for validating the functional impact of HSP gene mutations. For example:
• • SPAST knockout cells show impaired microtubule severing, confirming its role in axonal transport.
• • SPG11 knockout cells exhibit autophagic defects, linking the gene to autophagy.
• • These models allow researchers to perform complementation studies by reintroducing wild-type or mutant genes.

• • Isogenic pairs (wild-type vs. mutant) are used in high-throughput screening to identify compounds that rescue mutant phenotypes. For example:
• • Screening for drugs that enhance autophagy in SPG11 knockout cells.
• • Testing compounds that improve mitochondrial function in SPAST mutant cells.
• • Resistance modeling: Not applicable for HSP, but for cancer, similar approaches are used.

CRISPR synthetic lethality screens can identify genes that, when silenced, are lethal only in mutant cells. This can reveal potential therapeutic targets and biomarkers. For HSP, such screens may identify modifiers that exacerbate or alleviate the phenotype.