# Cerebrospinal fluid from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) with rapid evolution affects mitochondrial DNA transcription and network in human glioblastoma cell lines

Yago Cortes Pinheiro Gomes, Alice Bongers, Patricia Jeannin, Ana Carolina

Paulo Vicente, Antoine Gessain, Philippe V Afonso, Otavio Melo Espondola

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Yago Cortes Pinheiro Gomes, Alice Bongers, Patricia Jeannin, Ana Carolina Paulo Vicente, Antoine Gessain, et al.. Cerebrospinal fluid from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) with rapid evolution affects mitochondrial DNA transcription and network in human glioblastoma cell lines. 2025. pasteur-05124725

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1 Cerebrospinal fluid from patients with HTLV-1-associated myelopathy/tropical

2 spastic paraparesis (HAM/TSP) with rapid evolution affects mitochondrial DNA

3 transcription and network in human glioblastoma cell lines

4

5 **Short title:** CSF from HAM/TSP patients induces mitochondrial disturbances

6

7 Yago Côrtes Pinheiro Gomes¹,², Alice Bongers³, Patricia Jeannin³, Ana Carolina Paulo

8 Vicente², Antoine Gessain³, Philippe V. Afonso³*‡, Otavio Melo Espindola¹*‡

9 ¹ Instituto Nacional de Infectologia Evandro Chagas (INI), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

10

11 ² Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

12 ³ Unité Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur,

13 Université Paris Cité, Paris, France.

14

15 * Authors contributed equally

16 ‡ Corresponding authors:

17 PVA – Unité EPVO, Institut Pasteur, e-mail: pafonso@pasteur.fr;

18 OME – INI, Fiocruz, e-mail: otavio.espindola@ini.fiocruz.br

19

20 Author contributions:

21 Conceptualization: YCPG, PVA, OME; Formal Analysis: YCPG, AB, PVA, OME;

22 Funding Acquisition: PVA, OME; Investigation: YCPG, AB, PJ, OME; Resources: AG,

23 PVA, OME; Writing - Original draft preparation: YCPG, AB, PVA, OME; Writing -

24 Review & editing: ACPV, AG.
25 **ABSTRACT**

Human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive neurodegenerative disease that affects motor and sensory functions. While alterations in the cerebrospinal fluid (CSF) have been used to identify biomarkers for this disease, the effects of such modified CSF on neural cells remain unexplored. This study aimed to compare the effects of cerebrospinal fluid (CSF) from HTLV-1 asymptomatic carriers (HAC) with that from patients diagnosed with HAM/TSP on a glioblastoma cell line, U87-MG, which is often used to study neurodegenerative diseases. CSF samples were obtained from HTLV-1 asymptomatic carriers (HAC), and HAM/TSP patients categorized according to the speed of disease progression: very slow (HAMvs), typical (HAMt), and rapid (HAMr). RNA sequencing of U87-MG cells treated with CSF from HAMr patients revealed notable changes in gene expression with a significant downregulation of mitochondrial DNA transcripts. Confocal microscopy showed phenotypical changes in the mitochondrial network: glioblastoma cells exposed to HAMr CSF exhibited a less complex mitochondrial network compared to CSF from other patient groups, with longer mitochondrial branches, fewer junctions per branch length, and a slight increase in branch diameter. Despite these changes, U87-MG cells treated with the CSF from HTLV-1-infected donors with distinct neurological outcomes presented with similar mitochondrial metabolism. In conclusion, our findings suggest that CSF from HTLV-1-infected individuals induces mitochondrial alterations in glioblastoma cells, potentially helping to compensate for the cellular stress induced by CSF exposure. As mitochondrial alterations play an important role in other neurodegenerative diseases, we speculate that such alterations may participate in HAM/TSP pathogenesis.

49

50

51 **AUTHOR SUMMARY**

Human T-lymphotropic virus 1 (HTLV-1) is the etiological factor of a neurodegenerative disease named HTLV-1 associated myelopathy or tropical spastic paraparesis (HAM/TSP). While alterations in the cerebrospinal fluid (CSF) have been used to identify biomarkers for this disease, the impact of such an alteration on neural cells remains unexplored. In this study, we compared the impact of CSF from HTLV asymptomatic
carriers (HAC) and from HAM/TSP patients on a glioblastoma cell line, U87-MG, which
is often used to study neurodegenerative diseases. RNA sequencing analysis showed that
exposure to the CSF from HAM/TSP patients with rapidly evolving disease (HAMr) led
to a significant decrease of mitochondrial RNA expression. Glioblastoma cells treated
with HAMr CSF exhibited a less complex mitochondrial network. Despite these changes,
the metabolism of CSF-treated glioblastoma cells was unchanged, suggesting some
compensation in response to the cellular stress induced by CSF exposure. As
mitochondrial alterations play an important role in other neurodegenerative diseases, we
speculate that such alterations may participate in HAM/TSP pathogenesis.

66

67
68 INTRODUCTION

69

70 Human T-lymphotropic virus 1 (HTLV-1) is the etiological agent of a lymphoproliferative
disease, the adult T-cell leukemia/lymphoma (ATL), and a series of inflammatory
71
72 diseases, including a progressive neurodegenerative disease named HTLV-1 associated
73 myelopathy/tropical spastic paraparesis (HAM/TSP) (1-3).

74 HAM/TSP results from the infiltration of mononuclear cells, particularly HTLV-1-
75 infected CD4+ T cells and HTLV-1-specific cytotoxic CD8+ T cells, which mostly occurs
76 in the thoracic segment of the spinal cord. Consequently, local inflammation of the central
77 nervous system (CNS) is observed, leading to astrocyte activation, gliosis, demyelination
78 and neuronal loss, and ultimately spinal cord atrophy (4). Although the disease typically
79 progresses slowly, with mild symptoms presenting over several decades post-onset, a
80 rapid neurological decline can be observed in a proportion of HAM/TSP patients,
81 characterized by a severe loss of motor capability within approximately two years from
82 disease onset (5,6).

83 Consecutively, alterations in the CNS microenvironment are reflected by changes in the
84 composition of the cerebrospinal fluid (CSF), including the detection of proinflammatory
85 molecules such as CXCL10, chitotriosidase 1 (CHIT1), and neopterin (5,7). In a previous
86 work, we studied the composition of the CSF from HAM/TSP patients with varying
87 progression rates. Interestingly, those with rapid progression (HAMr) displayed a protein
88 profile associated with inflammatory processes, which was not observed in the other
89 groups (7).

90 This led to the use of CSF composition as biomarkers for the disease. We hypothesized
91 that changes in CSF composition could also directly affect the physiology of neural cells
92 and contribute to pathogenesis. Indeed, it has been reported that proinflammatory factors
93 such as interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), and CHIT1 can lead to
94 activation of microglia and other glial cell types (8–11).

95 Activated glial cells can trigger CNS inflammation, thereby significantly contributing to
96 the pathogenesis of neurodegenerative diseases such as Alzheimer's, Parkinson's, and
97 amyotrophic lateral sclerosis (ALS). Reactive phenotypes of astrocytes and microglia are
98 common features in the development of these neurodegenerative diseases (12).
Additionally, oxidative stress, an increased presence of reactive oxygen species (ROS), and disturbances in mitochondrial function are frequently observed (13). Notably, mitochondrial dysfunction can precede cell activation and neuroinflammation, culminating in neuronal death (14). The study of extracellular vesicles isolated from the plasma evidenced mitochondrial stress in HAM/TSP patients (15). However, no information is currently available regarding mitochondria in CNS cells in the context of HTLV-1 infection.

In this report, we aimed at comparing the impact of CSF from HTLV-1 asymptomatic carriers with CSF from HAM/TSP patients on glioblastoma cells – U87-MG cells which are often used to study neurodegenerative diseases. We showed that CSF from HAM/TSP patients with rapidly evolving disease induced changes in mitochondria transcriptomic, and mitochondrial structure. Intriguingly, these changes were not associated with any mitochondria related respiration change.

112

## 113 RESULTS

114

### 115 Transcriptional profile of glioblastoma cells incubated with CSF from HTLV-1-infected patients

117 Potential effects of the CSF from individuals infected with HTLV-1 on neural cells were initially tested by incubating glioblastoma U87-MG cell line with CSF for 6 and 24 hours.

118 Epidemiological characteristics of CSF donors is presented in Table 1. Due to volume limitations, CSF samples were pooled according to the speed of HAM/TSP progression, classified as very slow (HAMvs), typical (HAMt), or rapid (HAMr). Polled CSF from HTLV-1 asymptomatic carriers (HAC) were used as control. The concentrations of soluble factors from individual CSFs are presented in Fig S1. The values fall into the expected ranges. As expected, the concentrations of CXCL10 and neopterin increased with disease progression rate (16). CSF cells were eliminated at the time of collection by centrifugation.

127

128
After 6 hours, no significant difference was observed between cells stimulated with CSF from different HTLV-1 patient groups (Fig. S2). In contrast, principal component analysis (PCA) revealed that glioblastoma cells cultured with HAMr CSF exhibited significant changes after 24 hours of incubation compared to those treated with CSF from other patient groups (Fig. 1A). Hierarchical clustering analysis, as depicted in the heatmap, showed that the clustering was primarily attributed to the downregulation of mitochondrial genes (Fig. 1B), including MT-ATP6, MT-ATP8, MT-CO1, MT-CO2, MT-CO3, MT-CYB, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6.

138

<caption>Fig 1. Comparative transcriptomic analysis of U87-MG cells treated with CSF from HTLV-1-infected donors for 24 hours.</caption>

Bulk RNA sequencing results from triplicate samples of U87-MG cells treated for 24 hours with 25% (v/v) CSF pools obtained from HTLV-1-infected asymptomatic carriers (HAC), HAM/TSP patients categorized by progression rates—very slow (HAMvs), typical (HAMt), and rapid (HAMr). (A) Principal Component Analysis (PCA) was performed on all samples. (B) Heatmap shows the hierarchical clustering analysis of the samples (columns) and the top 100 genes (rows) with the highest variance among samples. Colors indicate transcripts that are more (red) or less (blue) expressed based on values normalized to their mean. (C) Scatterplot displays the log2 fold change from DESeq2 differential expression analysis between two comparisons: HAMr vs HAC and HAMr vs HAC+HAMvs+HAMt. The intersection of genes up- and downregulated in both analyses are highlighted in red and blue, respectively. (D) Bar plot representing the results of functional enrichment analysis, illustrating the biological processes and pathways associated with the downregulated genes identified in (C).

154

Relative expression analysis between cells incubated with HAMr CSF and with CSF from other patient groups showed that 12 mitochondrial genes exhibited the greatest reduction in expression among all 38 downregulated genes (Fig. 1C). Notably, only the gene encoding for the transcription factor ZBTB20 was found to be significantly overexpressed.

Accordingly, functional enrichment analysis of the common downregulated genes in HAMr CSF treated cells revealed that the altered processes/pathways were mostly associated with mitochondrial functions (Gene Ontology: oxidative phosphorylation, electron transport chain; Reactome: ATP synthesis) (Fig. 1D).
Collectively, our data demonstrate that CSF from HAMr patients induces mitochondrial stress, as evidenced by a marked downregulation of genes encoded by the mitochondrial genome in glioblastoma cells.

167

## 168 Changes in mitochondrial network in CSF-treated glioblastoma cells

To further investigate this apparent mitochondrial stress, we analyzed mitochondrial organization. U87-MG glioblastoma cells were stained with MitoTracker and incubated with CSF from HTLV-1-infected patients for 24 hours. Subsequently, the cells were fixed, observed by confocal imaging (Fig 2A), and analyzed using the Mitochondria Analyzer plugin in ImageJ. Data for HAMt CSF is not shown due to insufficient material for testing.

174

## 175 Fig 2. Changes in mitochondrial network organization following treatment with CSF from HTLV-1-infected donors.

176

U87-MG cells were treated for 24 hours with pooled CSF samples from HTLV-1 asymptomatic carriers (HAC) or from HAM/TSP patients with very slow (HAMvs) or rapid (HAMr) progression. The mitochondrial network was stained with MitoTracker DeepRed (red), and the nucleus with DAPI (blue). (A) Confocal images were obtained from five random fields from 2 independent experiments; representative images of field samples are shown. Scale bars on the merge image correspond to 20 µm. (B) Images were cropped to isolate single cells (number of cells analyzed = 19), and the mitochondrial network was analyzed using the Mitochondria Analyzer plugin in ImageJ software. Each point represents an image/cell, and the bar indicates the mean value. Differences between groups were evaluated by ANOVA followed by post-hoc analysis with t-test and Bonferroni correction for multiple comparisons. Significant differences are indicated as follows: \* p<0.05; \*\* p<0.01; \*\*\* p<0.001; \*\*\*\* p<0.0001; ns, non-significant.

189

When comparing glioblastoma cells treated with CSF from HAM/TSP patients to those treated with CSF from HAC donors (used as control), we found that mitochondria count remained constant.

We evaluated the morphology and complexity of the mitochondrial network by quantifying the diameter and mean branch length of the network, as well as the embranchment density.

The mean branch length was increased in cells treated with CSF from HAM/TSP patients, exhibiting a progressive increase from HAMvs to HAMr. The opposite trend was obtained
198 when considering the number of junction/branch length; the value was significantly lower
199 for HAMr, but the trend was also observed with HAMvs. These findings indicate that in
200 response to CSF from HAM/TSP patients the mitochondrial network undergoes
201 simplification and becomes more linear (Fig 2B). Notably, the network linearization was
202 accompanied by an increase in branch diameter.

203 Overall, we showed changes in the mitochondrial network induced by HAMr CSF,
204 characterized by a network simplification.
205

206 **Unchanged mitochondrial energy metabolism in glioblastoma cells incubated with**
207 **CSF from HTLV-1-infected patients**

208 We next investigated functional impact of CSF on glioblastoma cells. Cell proliferation
209 was monitored for 24 hours on IncuCyte SX5 using HD phase contrast imaging. The
210 slopes of the curves were identical, meaning that U87-MG cell confluence evolved
211 similarly across the various conditions (Fig. S3). This suggests that no CSF altered
212 specifically cell toxicity and proliferation.

213 To determine whether transcriptional and structural changes in mitochondria of U87-MG
214 glioblastoma cells affected mitochondrial energy production, cells were incubated for 24
215 hours with CSF from HTLV-1-infected individuals, and the oxygen consumption rate
216 (OCR) was assessed using the Seahorse technology (Fig. 3A). We found that there is no
217 significant difference in mitochondria-related functions. Indeed, basal respiration (A),
218 maximal respiration (B), spare respiration capacity (C), ATP-linked respiration (D) and
219 proton link (E) were comparable in U87-MG glioblastoma cells treated with the different
220 CSF. Of note, non-mitochondrial oxygen consumption (G) was decreased in HAM CSF
221 treated cells compared to HAC CSF.
222

223 **Fig 3. Mitochondrial function of glioblastoma cells treated with CSF from HTLV-1-**
224 **infected donors.**

225 U87-MG cells were incubated for 24 hours in the presence of 25% (v/v) pooled CSF
226 samples from HTLV-1 asymptomatic carriers (HAC), and HAM/TSP patients with very
227 slow (HAMvs), typical (HAMt), and rapid (HAMr) progressions in complete DMEM.
228 (A) The oxygen consumption rate (OCR) was assessed with the Seahorse XF Cell Mito
229 Stress Test kit on a Seahorse XFe96 Analyzer (Agilent) after injection (at the final
230 concentration) of 1.5 µM oligomycin – an ATP synthase inhibitor–, 1µM FCCP - a strong
231 coupler of oxidative phosphorylation, and 0.5µM rotenone/antimycin A (Rot/AA) -
232 inhibitors of respiratory complexes I and III. OCR were normalized but the number of 233 cells per well (Draq5 staining post treatment). The differences in OCR upon sequential 234 allow to calculate: (B) basal respiration, (C) maximal respiratory capacity, (D) spare 235 respiratory capacity, (E) ATP-linked respiration (F) proton leak and (G) non 236 mitochondrial respiration. Each point represents a replicate (n=4 to 6 replicates, 237 depending on the availability of CSF volumes) and mean and standard deviation are 238 indicated. Differences between groups were evaluated by ANOVA followed by post-hoc 239 analysis with Dunnett correction for multiple comparisons. Significant differences are 240 indicated as follows: * p<0.05; ** p < 0.01.

241

242 In summary, our data indicate that although HAMr CSF treatment induces cellular stress 243 and alters the mitochondrial network, cells adapt in a way that preserves mitochondrial 244 function.

245

246 DISCUSSION

247 Until now, CSF composition has been used as a biomarker of HTLV-1-associated 248 neurodegenerative disease. However, since CSF from HAM/TSP patients can be enriched 249 in inflammatory elements (e.g. CHIT1 (7), CXCL10 and metabolites such as neopterin 250 (16,17), as well as exosomes (15)), we speculated that it may have an impact on the 251 biology of neural cells and may participate in pathogenesis. This study represents the first 252 investigation into the impact of diseased CSF on neural cells.

253 The potential effect of CSF from individuals infected with HTLV-1 on CNS cells was 254 evaluated on the U87-MG glioblastoma cell line, a model commonly employed in *in vitro* 255 studies of neurodegenerative diseases (18-20).

256 We found that CSF from HAMr patients induces changes in the transcriptome of 257 glioblastoma cells, and changes in the mitochondrial network, when compared to cells 258 treated with CSF from HAC donors. However, the overall mitochondrial metabolism 259 remained largely unchanged, at least when cells were treated for 24h with the different 260 CSF pools from patients with different neurological status. As mitochondrial changes 261 have been reported in other neurodegenerative diseases, and participate in pathogenesis 262 of ALS, MS and Parkinson's, we assume that during HAM/TSP pathogenesis the 263 alterations in CSF composition may alter the neural functions and participate in the 264 disease.
265 Mitochondrial stress is recognized as a common feature of neurodegenerative diseases, 266 including Alzheimer's, Parkinson's, and ALS (21,22). During oxidative phosphorylation, 267 reactive oxygen species (ROS) are produced by mitochondria. Under pathological 268 conditions, ROS can damage mtDNA, which is more sensitive than nuclear DNA (23). 269 Consistent with this, upregulation of ZBTB20 gene expression was observed in cells 270 exposed to HAMr CSF. This gene encodes a protein known to play a central role in 271 inducing oxidative stress, ROS production, mitochondrial fission, and further 272 inflammation in ox-LDL-stimulated macrophages (24).

273 Mitochondrial network alterations are frequently reported in neurodegenerative diseases 274 (25). In Alzheimer's disease (26), ALS (27), and Parkinson's syndrome (28), mitochondria 275 in neural cells often undergo fragmentation. In glioblastoma cells treated with CSF from 276 HAMr patients, we revealed that mitochondria undergo network simplification instead, 277 with a decrease in the number of junctions per branch length.

278 Moreover, cells exposed to HAMr CSF displayed an increase in both branch length and 279 diameter of the mitochondrial network. This evidence, combined with the lack of change 280 in mitochondria numbers, suggests a moderate enlargement of this organelle (29). Since 281 both swelling and fission can result in a spherical-shaped mitochondria (30), it is likely 282 that both processes are present under this condition. In contrast, no differences were 283 observed in the expression of genes encoding proteins responsible for mitochondrial 284 fusion, such as MFN1, MFN2, or OPA1, or fission, including DNM1L (DRP1 protein), 285 MFF, or FIS1. This suggests that factors present in HAMr CSF might trigger 286 mitochondrial morphological changes through mechanisms other than modulation of 287 gene expression, such as protein-protein interactions or ionic imbalances. Moreover, cells 288 incubated with HAMvs CSF also displayed mitochondria with longer branches and 289 increased diameter compared to those treated with HAC CSF, although these changes 290 were less pronounced than those observed with HAMr CSF.

291 Although identifying the specific factors responsible for mitochondrial stress would have 292 been valuable in the context of this study, previous research has demonstrated that protein 293 alterations in the CSF of HAM/TSP patients are highly diverse. Increased levels of 294 CXCL10 and CHIT1, as well as alterations on metabolites or exosome levels may impact 295 mitochondrial physiology either individually or synergically. Given the wide range of 296 potential toxic elements to mitochondria, we did not assess them individually in this work.
297 Surprisingly, despite changes in markers of mitochondrial stress (transcriptome and network organization), the overall energy metabolism in CSF-treated glioblastoma cells 298 remained unaffected. This suggests the activation of compensatory mechanisms. Whether 299 this response is specific to the glioblastoma cell line used, or a general ability of cells to 300 adapt to CSF-induced stress remains to be determined. 301

302
303 This study had limitations. Firstly, the small sample size and limited CSF volume 304 available for all assays led us to work on CSF pools. Nevertheless, the long-term 305 conduction of cohort studies with structured neurological follow-up to determine 306 HAM/TSP progression rate is quite challenging and is acknowledged as a strength of this 307 study. 308

308 Further studies will have to be performed with CSF samples from individual patients. We 309 also acknowledge the lack of “normal CSF control” from non-HTLV-1-infected patients. 310 It remains unknown whether CSF form uninfected individuals could lead to 311 mitochondrial network alterations comparable to those induced by CSF from HTLV-1 312 asymptomatic carriers. These were impossible to obtain due to ethical reasons. In some 313 articles, control CSF corresponds to CSF from patients with other neurological diseases 314 or diseases with unknown origin, which raises the issue of the nature of the sample: should 315 it be considered as a positive or negative control. Therefore, the present study focuses 316 primarily on the comparison of CSF from HAC and CSF from HAM patients -which is 317 to the best of our knowledge the best control. 318

318
319 Secondly, experiments will have to be performed on other cell types that may represent 320 more accurately neural resident cells such as astrocytes, microglia and neurons, which 321 will be the focus of follow-up studies. 322

321 Finally, in this study, we focused on the cellular response following a 24-hour treatment, 322 as this time point revealed both transcriptomic and morphological changes. However, 323 considering that HAM/TSP is a chronic condition, future investigations will need to 324 explore longer treatment durations to assess whether extended exposure affects 325 mitochondrial function or triggers mitophagy. Notably, our previous work demonstrated 326 signs of mitochondrial stress in extracellular vesicles from the plasma of HAM/TSP 327 patients (15). Damaged mitochondria can also be released by activated microglia and 328 astrocytes, potentially contributing to neuronal damage (31). This mechanism could play
a role in the accelerated neurological decline observed in rapid HAM/TSP progressors
and warrants further investigation.

Despite these limitations, our results suggest that CSF from HAM/TSP patients induces
cellular stress, which appears to be compensated by alterations in the mitochondrial
network, particularly in cases of rapidly evolving HAM/TSP.

# MATERIAL AND METHODS

## Study design, population, and ethics statement

CSF samples originated from a cohort of HTLV-1-infected individuals who were followed
at the Evandro Chagas National Institute of Infectious Diseases (INI) of the Oswaldo Cruz
Foundation (FIOCRUZ) in Rio de Janeiro, Brazil (16,32).

HTLV-1-infected patients were clinically categorized as asymptomatic carriers (HAC,
n=11) or as having HAM/TSP (n=20) as previously described (16,32). Neurological
involvement was evaluated by the IDS disability scale (33). The speed of HAM/TSP
progression was determined by an index calculated as the quotient of the IDS score and
the disease duration, defined as the time elapsed from disease onset to CSF withdrawal.
Based on this index, HAM/TSP progression was classified as follows: ≤ 0.37 points/year
as very slow (HAMvs, n=6), between 0.38 and 1.44 points/year as typical (HAMt, n=9),
and ≥ 1.45 points/year as rapid, HAMr, n=5). CSF cells were removed at the time of
collection by centrifugation.

Characteristics of donors and individual samples are presented in table 1 and
supplementary figure 1.

## Cell line and culture conditions

The U87-MG (ATCC HTB-14) glioblastoma cell line was cultured in complete DMEM
(supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and
100 µg/mL streptomycin) at 37°C in a 5% CO₂ atmosphere.

## RNA Sequencing and analysis

U87-MG cells were cultured in triplicate at 2×10⁵ cells/well in 24-well plates in 500 µl of complete DMEM with 25% (v/v) pooled CSF from patients categorized as HAC,
358 HAMvs, HAMt, or HAMr, with untreated cells serving as a negative control. After
359 incubation for 6 and 24 hours at 37°C in 5% CO₂ atmosphere, total RNA was extracted
360 using the PureLink RNA Micro Scale kit (Invitrogen, USA), following the manufacturer's
361 instructions.

362 RNA quality and concentration were determined using the RNA ScreenTape kit on a
363 TapeStation system (Agilent). Paired-end genomic libraries of enriched mRNAs were
364 constructed with the Stranded mRNA Prep kit (Illumina) and the Illumina RNA UDI A
365 LIG 96 indexes (IDT Technologies), according to the manufacturer's instructions.
366 Sequencing was performed on an Illumina P3 cell-flow for 200 cycles on a NextSeq 2000
367 system (Illumina). Alignment of reads was performed using STAR v2.7.5c software (34),
368 with the human genome reference version GRCh38.p14 (35).

369 Differential gene expression was calculated with DESeq2 (36). A log₂ fold-change > 0.5
370 or < -0.5 and adjusted *p*-values < 0.05 were considered significant. Functional enrichment
371 analysis was performed using clusterProfile (37). The data that support the findings of
372 this study are publicly available from Gene Expression Omnibus database with the
373 identifier GSE290808.

374 **Mitochondrial network analysis**

375 U87-MG cells were cultured on coverslips (10⁵ cells/cm²) and stained with MitoTracker
376 Deep Red (Invitrogen) following the manufacturer's instructions. Cells were treated with
377 CSF (25% v/v). After 24 hours, cells were fixed and confocal images were obtained using
378 a Zeiss LSM 700 microscope. Images were cropped to isolate single cells and analyzed
379 using ImageJ/Fiji software with the Mitochondria Analyzer plugin (38).

380 **Characterization of mitochondrial energy metabolism**

381 To assess the effect of CSF from HTLV-1-infected donors on mitochondrial function,
382 U87-MG cells (12,000 cells/well) were seeded and cultured in XFe96 microplates
383 (Agilent) with complete DMEM (50 µL/well). After 24 hours, the culture medium was
384 replaced with 25% (v/v) CSF in complete DMEM, and cells were incubated for an
385 additional 24 hours. The proliferation of glioblastoma cells was monitored for 24 hours
386 using an IncuCyte SX5 with HD phase contrast imaging.

387 Mitochondrial function was assessed using the Seahorse XF Cell Mito Stress Test and the
388 Seahorse XFe96 FluxPak on a Seahorse XFe96 Analyzer (Agilent) under hypoxic
conditions, following the manufacturer's instructions. Briefly, cell supernatants were removed and 180 µL of pre-warmed Seahorse XF DMEM supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine (Agilent) were added per well. Microplates were incubated in a CO₂-free incubator at 37°C for 1 hour. The assay was performed by measuring the OCR after injection (at the final concentration) of 1.5 µM oligomycin, 1 µM FCCP, and 0.5 µM rotenone/antimycin A. CSF pools were tested at least in quadruplicate, and OCR values were normalized by cell density determined at the end of the experiment following cellular staining with Draq5.

## 397 Statistical analysis

Gene expression levels are shown in volcano plots and differences were evaluated by FDR test. Gene expression was also evaluated by principal component analysis (PCA) and clustering analysis, showing the top 100 transcripts displaying the highest variance shown in heatmap graphs. Functional enrichment analysis was carried out with clusterProfile package. Alterations in mitochondrial function parameters were evaluated between groups by ANOVA, followed by post-hoc analysis with t-test and Bonferroni correction for multiple comparisons. Differences were considered statistically significant when $p < 0.05$.

## 406 Ethics Statement

The study protocol (CAAE 27057119.9.0000.5262) was approved by the Institute National de Infectiologia Evandro Chagas/Fundação Oswaldo Cruz (INI/FIOCRUZ) Research Ethics Committee on February 6, 2020, and by the Brazilian National Commission of Ethics in Research (CONEP) on March 7, 2023, and written informed consent was obtained from all participants.

## 412 Funding

The study was funded by the Programa Jovens Pesquisadores - INI/FIOCRUZ (OME, Grant No. INI-003-FIO-19-2-14), ANRS MIE (PVA, Grant No. 24150), Institut Pasteur - Programme Transversal de Recherche (PTR) (PVA, Grant No. 1786-24), and Investissement d'Avenir/Laboratoire d'Excellence (LabEx)/Integrative Biology of Emerging Infectious Diseases (AG, Grant No. ANR10-LBX-62 IBEID). OME and YCG were supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil; AB was supported by Ministère de la Recherche, France. The funders
420 had no role in study design, data collection and analysis, decision to publish, or
421 preparation of the manuscript.

422 Acknowledgements

423 Seahorse experiments were conducted under the supervision of Samy Dufour, François
424 Le Gourroue and Milena Hasan of the UtechS Single Cell Biomarkers; confocal
425 microscopy was performed at UtechS PBI/Imagery platform at Institut Pasteur. NGS and
426 confocal microscopy were performed at the NGS (P01-010) and Microscopy (P05-001)
427 facilities of the FIOCRUZ's Network of Technological Platforms.

428

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<table><thead><tr><th>Clinical status</th><th>Sex</th><th>Age (years)</th><th>HTLV-1 PVL (% PBMC)</th><th>EIPEC-2 scale (0-31 points)</th><th>Disability</th></tr></thead><tbody><tr><td>HAC</td><td>F</td><td>64,71</td><td>9,12</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>M</td><td>54,17</td><td>3,15</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>F</td><td>50,70</td><td>10,05</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>F</td><td>67,99</td><td>6,46</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>F</td><td>60,32</td><td>4,45</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>F</td><td>72,75</td><td>0,37</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>M</td><td>75,55</td><td>2,83</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>F</td><td>39,08</td><td>3,89</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>M</td><td>68,03</td><td>10,91</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>M</td><td>59,50</td><td>0,51</td><td>0</td><td>No</td></tr><tr><td>HAC</td><td>F</td><td>72,89</td><td>4,58</td><td>0</td><td>No</td></tr><tr><td>HAMvs</td><td>M</td><td>65,47</td><td>6,21</td><td>9</td><td>Mild</td></tr><tr><td>HAMvs</td><td>F</td><td>54,18</td><td>4,82</td><td>4</td><td>Mild</td></tr><tr><td>HAMvs</td><td>F</td><td>34,42</td><td>5,96</td><td>5</td><td>Mild</td></tr><tr><td>HAMvs</td><td>M</td><td>59,53</td><td>16,93</td><td>5</td><td>Mild</td></tr><tr><td>HAMvs</td><td>M</td><td>52,49</td><td>26,42</td><td>9</td><td>Mild</td></tr><tr><td>HAMvs</td><td>F</td><td>59,54</td><td>9,38</td><td>2</td><td>Mild</td></tr><tr><td>HAMt</td><td>F</td><td>54,39</td><td>13,96</td><td>8</td><td>Mild</td></tr><tr><td>HAMt</td><td>M</td><td>61,13</td><td>11,09</td><td>20</td><td>Moderate</td></tr><tr><td>HAMt</td><td>F</td><td>50,11</td><td>2,68</td><td>9</td><td>Mild</td></tr><tr><td>HAMt</td><td>F</td><td>67,81</td><td>5,40</td><td>22</td><td>Severe</td></tr><tr><td>HAMt</td><td>F</td><td>68,11</td><td>2,72</td><td>18</td><td>Moderate</td></tr><tr><td>HAMt</td><td>M</td><td>28,81</td><td>10,73</td><td>13</td><td>Moderate</td></tr><tr><td>HAMt</td><td>F</td><td>43,46</td><td>3,34</td><td>9</td><td>Mild</td></tr><tr><td>HAMt</td><td>F</td><td>82,43</td><td>3,00</td><td>9</td><td>Mild</td></tr><tr><td>HAMt</td><td>F</td><td>48,35</td><td>16,01</td><td>22</td><td>Severe</td></tr><tr><td>HAMr</td><td>M</td><td>55,38</td><td>10,30</td><td>3</td><td>Mild</td></tr><tr><td>HAMr</td><td>M</td><td>58,18</td><td>1,25</td><td>8</td><td>Mild</td></tr><tr><td>HAMr</td><td>F</td><td>61,31</td><td>0,60</td><td>3</td><td>Mild</td></tr><tr><td>HAMr</td><td>M</td><td>60,39</td><td>4,45</td><td>14</td><td>Moderate</td></tr><tr><td>HAMr</td><td>F</td><td>28,00</td><td>8,79</td><td>16</td><td>Moderate</td></tr></tbody></table>

558

559

<caption>Table 1 – Description of the CSF donors</caption>

560

561

For each individual CSF sample, clinical status, age, sex, proviral load and disease characteristics are presented.

562
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>
1 SUPPORTING INFORMATION

2 Cerebrospinal fluid from patients with HTLV-1-associated myelopathy/tropical
3 spastic paraparesis (HAM/TSP) with rapid evolution affects mitochondrial DNA
4 transcription and network in human glioblastoma cell lines

5 Yago Côrtes Pinheiro Gomes¹,², Alice Bongers³, Patricia Jeannin³, Ana Carolina Paulo

6 Vicente², Antoine Gessain³, Philippe V. Afonso³*‡, Otavio Melo Espindola¹*‡

7

<figure><img src="image_18.png" /></figure>

8

<caption>9 Fig S1 – Composition of the CSF samples used in the study.</caption>

10 The concentrations of 20 detectable soluble factors are shown for each individual sample
11 based on clinical status. The bar indicates the mean value, representing the concentration
12 in the CSF pools used in the study. Of note, CSF pools did not contain any cell as they
13 were centrifuged at the time of collection. Differences were assessed using ANOVA,
14 followed by Tukey's post-hoc test. Significant differences are indicated as follows: \* p<
15 0.05; \*\* p < 0.01.
CSF from HAM/TSP patients induces mitochondrial disturbances

<figure><img src="image_19.png" /><figcaption>Fig S2. Principal Component analysis (PCA) of bulk RNA-seq of U87-MG cells treated with CSF for 6 hours.</figcaption></figure>

Principal Component Analysis (PCA) results from triplicate or duplicate samples of U87-MG cells treated for 24 hours with 25% (v/v) pooled CSF from HTLV-1 asymptomatic patients (HAC, n=3), and HAM/TSP patients categorized as very slow (HAMvs, n=3), typical (HAMt, n=3), and rapid (HAMr, n=2) progression.
25

<figure><img src="image_20.png" /></figure>

26

27

<caption>Fig S3. CSF treatment induces no change in glioblastoma cell proliferation.</caption>

28

29

30

31

32

U87-MG cells were treated for 24 hours with 25% (v/v) pooled CSF samples from HTLV-1 asymptomatic carriers (HAC), and HAM/TSP patients with very slow (HAMvs), typical (HAMt), and rapid (HAMr) progressions. Cell proliferation was analyzed according to monolayer confluence in an IncuCyte SX5 using HD phase contrast imaging. Data are shown as mean, standard deviation (n>4 replicates).

33