ALS-FTD-linked CCNFS621G drives increased hippocampal astrocyte ramification and mitochondrial dysfunction and impairs motor neuron excitability.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with overlapping pathology. Mutations in CCNF, encoding the E3 ubiquitin ligase, Cyclin F, can cause ALS, FTD, or both, even within the same family. Most prior studies of CCNFS621G have relied on overexpression systems, potentially confounding outcomes through disruption of endogenous Cyclin F. Here, we generated the first knock-in mouse model of endogenous CcnfS621G using CRISPR/Cas9. Heterozygous and homozygous CcnfS621G mice showed no motor decline or neuronal loss after 18 months, however immunohistochemistry revealed increased hippocampal astrocyte ramification, with sex-, age, and subfield-dependent effects. These data indicate that endogenous CcnfS621G may prime early astrocyte alterations in the absence of overt neurodegeneration. Similar astrocyte morphological changes were observed in canonically affected regions of sporadic ALS and FTD-ALS patients post mortem, as well as in CCNFS621G iPSC-derived astrocytes following inflammatory stimulation. Proteomics on Ccnf mice identified early dysregulation of pathways related to translation, mitochondrial function, cytoskeletal remodelling, synaptic transmission and neuroinflammation. Correspondingly, CCNFS621G iPSC-derived astrocytes displayed impaired mitochondrial membrane potential and altered network morphology under both basal and inflammatory stimuli. As altered neuronal excitability is a hallmark of ALS, we examined astrocyte-driven changes to neuronal excitability. CCNFS621G iPSC-derived motor neurons cultured alone were hyperexcitable, firing more action potentials than isogenic controls. Remarkably, co-culture with CCNFS621G astrocytes, but not isogenic control astrocytes, abolished repetitive firing, increased the proportion of neurons unable to generate action potentials, and reduced voltage-gated sodium currents in CCNFS621G and isogenic control neurons. Together, these findings identify astrocyte alterations as an early feature of CCNFS621G-mediated disease, in the absence of neuronal loss. Moreover, the combination of astrocytic mitochondrial dysfunction and the ability of CCNFS621G astrocytes to suppress repetitive neuronal firing suggests a critical astrocyte-driven non-cell autonomous mechanism that may contribute to an oligogenic role for CCNF in ALS/FTD pathogenesis.