Towards High-Accuracy Athletic Injury Predictions Using a First-Principles Modelling Approach: Theory to (Future) Practice.

Approximately one-third of all genes in the eukaryotic genome are predicted to encode transmembrane proteins that must traffic through various cellular compartments to function effectively (, ). Defective transport of these proteins has been increasingly linked to a range of neurological diseases, including frontotemporal dementia, Charcot-Marie-Tooth disease, and several types of hereditary spastic paraplegia (HSP) (, ). Among these, adaptor protein complex 4–associated (AP-4–associated) hereditary spastic paraplegia (AP-4-HSP) has emerged as a prototypical disorder of impaired protein trafficking. Clinically, AP-4-HSP manifests in early childhood with developmental delay/intellectual disability, progressive spasticity leading to loss of ambulation, epilepsy, secondary microcephaly, and developmental brain malformations (–). The condition arises from biallelic loss-of-function variants in one of four genes (, , , and ) encoding the subunits of the heterotetrameric AP-4 complex, which is critical for sorting cargo proteins from the -Golgi network (TGN) to early and late endosomes and pre-autophagosomal structures (, ). Recent studies have identified autophagy-related protein 9A (ATG9A), the sole transmembrane protein in the autophagic machinery, as an important cargo of AP-4 (–). In AP-4–deficient cells, ATG9A accumulates in the TGN and is depleted from peripheral compartments. In neurons, this mislocalization leads to deficits in autophagy, impaired axon maintenance, and length-dependent axonal degeneration (–). However, residual autophagic activity and the presence of ATG9A outside the TGN suggest an alternative, AP-4–independent export mechanism (, ). This raises critical questions about how ATG9A trafficking is regulated in the context of AP-4 deficiency. Genome-wide functional genomics screens have emerged as powerful tools to uncover regulators of cellular signaling. However, traditional approaches such as loss-of-function screening using small interfering RNA (siRNA) libraries are often limited by poor reproducibility due to inconsistent gene knockdown and off-target effects (, ). Advances in CRISPR/Cas9–based gene interference, particularly its application in high-throughput screening assays, have addressed many of these limitations, ushering in a new era in functional genomics (). While several large-scale CRISPR screens have been successfully conducted in recent years — primarily as pooled screens in which cells are bulk-transfected with a single-guide RNA (sgRNA) library, selected for specific phenotypes, and analyzed using next-generation sequencing — these approaches pose significant challenges for assessing complex cellular phenotypes (, ).