# Multi-omics-based phenotyping of AFG3L2-mutant lymphoblasts determines key factors of a pathophysiological interplay between mitochondrial vulnerability and neurodegeneration in spastic ataxia type 5

## TL;DR

This study uses multi-omics to explore how AFG3L2 mutations in spastic ataxia type 5 disrupt mitochondrial and cellular functions, leading to neurodegeneration.

## Contribution

The study reveals novel pathophysiological mechanisms in SPAX5 through multi-omics analysis of AFG3L2-mutant lymphoblasts.

## Key findings

- AFG3L2 mutations disrupt mitochondrial dynamics, calcium homeostasis, and lipid metabolism.
- Loss of AFG3L2 function leads to cytoskeletal instability and respiratory dysfunction in SPAX5.
- Multi-omics analysis identifies systemic cellular disturbances contributing to neurodegeneration.

## Abstract

Mitochondrial integrity is fundamental to cellular function, upheld by a network of proteases that regulate proteostasis and mitochondrial dynamics. Among these proteases, AFG3L2 is critical due to its roles in maintaining mitochondrial homeostasis, regulating mitochondrial protein quality, and facilitating mitochondrial biogenesis. Mutations in AFG3L2 are implicated in a spectrum of diseases, including spinocerebellar ataxia type 28 (SCA28) and spastic ataxia 5 (SPAX5), as well as other systemic conditions. This study employs a multi-omics approach to investigate the biochemical impact of AFG3L2 mutations in immortalized lymphoblastoid cell lines derived from a patient with biallelic variants leading to spastic ataxia (SPAX5). Our proteomic analysis revealed AFG3L2 impairment, with significant dysregulation of proteins critical for mitochondrial function, cytoskeletal integrity, and cellular metabolism. Specifically, disruptions were observed in mitochondrial dynamics and calcium homeostasis, alongside downregulation of key proteins like COX11, a copper chaperone for complex IV assembly, and NFU1, an iron-sulfur cluster protein linked to spastic paraparesis and infection-related worsening. Lipidomic analysis highlighted substantial alterations in lipid composition, with significant decreases in sphingomyelins, phosphatidylethanolamine, and phosphatidylcholine, reflecting disruptions in lipid metabolism and membrane integrity. Metabolomic profiling did not reveal any significant findings. Our comprehensive investigation into loss of functional AFG3L2 elucidates a pathophysiology extending beyond mitochondrial proteostasis, implicating a wide array of cellular processes. The findings reveal substantial cellular disturbances at multiple levels, contributing to neurodegeneration through disrupted mitochondrial respiratory chain, calcium homeostasis, cytoskeletal integrity, and altered lipid homeostasis. This study underscores the complexity of SPAX5 pathophysiology and the importance of multi-omics approaches in developing effective strategies to address the impact of loss of functional AFG3L2. Our data also highlight the value of immortalized lymphoblastoid cells as a tool for pre-clinical testing and research, offering a detailed biochemical fingerprint that enhances our understanding of SPAX5 and identifies potential areas for further investigation.

Pathophysiological impact of AFG3L2 mutations in SPAX5 as revealed by multi-omics analysis. The mitochondrial protease AFG3L2 is localized in the inner mitochondrial membrane (IMM), where it plays a critical role in maintaining mitochondrial proteostasis. Its physiological functions include ATP-dependent protein hydrolysis, calcium homeostasis, and the degradation of misfolded proteins, which are essential for mitochondrial integrity, axonal development, and neuronal health. Mutations in AFG3L2 impair these functions, leading to dysregulation of neuronal function-related proteins, cytoskeletal instability, and respiratory dysfunction. These molecular disruptions result in neurodegenerative phenotypes, including SPAX5 (spastic ataxia 5), SCA28 (spinocerebellar ataxia 28), and OPA12 (optic atrophy 12). On the right, findings from a patient with SPAX5 are summarized. Multi-omics analysis of lymphoblastoid cells revealed mitochondrial calcium homeostasis disruption, impaired lipid metabolism, and immune pathway activation. These cellular disturbances highlight a systemic impact of AFG3L2 dysfunction, contributing to the neurodegenerative features observed in SPAX5. Collectively, these findings underline the critical role of AFG3L2 in cellular homeostasis and emphasize the value of multi-omics approaches in understanding the pathophysiology of SPAX5. Created using BioRender.com.