The parasitic protozoa Toxoplasma gondii chronically infects the central nervous system of an estimated one-third of the human population. Infection is generally subclinical, but immunocompromised individuals can experience a variety of neurological symptoms. Meta-analyses of T. gondii seropositivity have suggested a correlation between T. gondii infection and neurologic disease. While mechanistic studies on the relationship between T. gondii infection and neurologic disease have been attempted in mice, mice are particularly susceptible to T. gondii , making them an effective model for investigating mechanisms of infection, but not ideal for examining the relationship between long-term chronic T. gondii infection and neurologic disease. Rats more closely mimic human clearance of T. gondii after acute infection, but a lack of rat models of neurologic disease has limited studies on the interplay between T. gondii infection and neurologic disease progression. We have employed a previously characterized rat model of a complex form of hereditary spastic paraplegia (HSP), a class of neurodegenerative disorders which cause axonal degeneration and lower limb spasticity, in order to assess the effect of chronic T. gondii infection on neurodegenerative disease. We find that infected rats with hereditary spastic paraplegia exhibit significantly exacerbated behavioral and neuromorphological HSP symptoms compared to uninfected HSP mutant rats, with little correlative effect in infected versus uninfected control animals. We further find that all infected rats regardless of genotype exhibit a robust immune response to T. gondii infection, effectively clearing the parasite below the limit of detection of multiple assays of parasitemia and exhibiting no detectable increase in neuroinflammation seven weeks post-infection. These results suggest that chronic undetected T. gondii infection may exacerbate neurodegenerative disease even in immunocompetent individuals and may contribute to neurodegenerative disease heterogeneity.Author Summary The long-term consequences of previous acute infections are poorly understood, but are becoming increasingly appreciated, particularly in the era of long Covid. Altered progression of other diseases later in life may be among the long-term consequences of previous infections. Here we investigate the relationship between previous infection with the parasite Toxoplasma gondii , which infects ∼30% of the global population, and neurodegenerative disease using a rat model of hereditary spastic paraplegia (HSP). We find that previous infection with T. gondii accelerates motor dysfunction in HSP rats, despite robust clearance of the parasite by infected rats. Our results suggest that previously cleared infections may alter the progression of other diseases later in life and contribute to neurodegenerative disease heterogeneity.### Competing Interest StatementThe authors have declared no competing interest.

Inherited forms of motor neuron disease (MND), including hereditary spastic paraplegias (HSP), are associated with the death or dysfunction of nerve cells that control skeletal muscle activity. However, in some cases, the impacts of genetic variants underlying MND act in a non-cell autonomous manner, instead affecting the function of other cell types necessary for neuronal maintenance. Pathological mutations in TFG, which have been implicated in HSP, lead to axonopathy within the corticospinal tract, but it remains unclear whether this problem arises due to perturbations within neurons or supporting neuroglia. To address this question, we leveraged a rat model harboring the recessive TFG p.R106C mutation (mRATBN7.2, g.11:43897639C>T, c.316C>T), which recapitulates multiple phenotypes associated with HSP in humans, including progressive motor deficits, leg spasticity, and indications of an inflammatory response within the motor cortex. In particular, we took advantage of cell type-specific gene therapies to demonstrate that the reintroduction of wild-type TFG into synapsin 1-positive neurons provides robust protection against MND, whereas its expression in GFAP-positive glial cells provides no significant improvement in quantitative measures of gait, despite a dramatic reduction in the presence of reactive astrocytes throughout the brain. These data strongly suggest that therapeutic approaches targeting neurons should be pursued in cases of TFG-HSP, with our animal model offering a unique platform for preclinical assessment.

Pathological variants in Trk-fused gene (TFG) have been implicated in a variety of neurodegenerative conditions. In particular, mutations within its amino-terminal PB1 domain have been suggested to cause hereditary spastic paraplegia (HSP), resulting in progressive lower limb spasticity and weakness. The structural basis for this effect is unknown. Here, we combine X-ray crystallography and cryo-electron microscopy to determine a structural model of TFG, demonstrating the mechanism by which it forms octameric ring complexes. A network of electrostatic and hydrophobic interactions defines the interface between protomers. Moreover, we show that mutations identified previously in HSP patients disrupt this interface, destabilizing octamers, which ultimately leads to axonopathy. Surprisingly, the impacts of these variants are not equivalent in vivo, highlighting the existence of multiple, distinct mechanisms by which TFG mutations contribute to neurodegenerative disease.

Molecular pathways that intrinsically regulate neuronal maintenance are poorly under-stood, but rare pathogenic mutations that underlie neurodegenerative disease can offer important insights into the mechanisms that facilitate lifelong neuronal function. Here, we leverage a rat model to demonstrate directly that the TFG p.R106C variant impli-cated previously in complicated forms of hereditary spastic paraplegia (HSP) underlies progressive spastic paraparesis with accompanying ventriculomegaly and thinning of the corpus callosum, consistent with disease phenotypes identified in adolescent patients. Analyses of primary cortical neurons obtained from CRISPR-Cas9-edited ani-mals reveal a kinetic delay in biosynthetic secretory protein transport from the endo-plasmic reticulum (ER), in agreement with prior induced pluripotent stem cell-based studies. Moreover, we identify an unexpected role for TFG in the trafficking of Rab4A-positive recycling endosomes specifically within axons and dendrites. Impaired TFG function compromises the transport of at least a subset of endosomal cargoes, which we show results in down-regulated inhibitory receptor signaling that may contribute to excitation-inhibition imbalances. In contrast, the morphology and trafficking of other organelles, including mitochondria and lysosomes, are unaffected by the TFG p.R106C mutation. Our findings demonstrate a multifaceted role for TFG in secretory and endo-somal protein sorting that is unique to cells of the central nervous system and highlight the importance of these pathways to maintenance of corticospinal tract motor neurons.