WO2025078851A1 - Methods of treating cognitive deficit - Google Patents

Abstract

Here, the Inventors show that chronic IL- 1 -induced H2A.X signaling, which mediated DNA DSB response, in hippocampal neurons is a critical driver of cognitive deficits in chronic low grade neuroinflammation. They show that mice chronically infected with the brain- persisting T. gondii parasite display deficits in spatial memory consolidation without general memory loss. Extensive mapping of the neuroinflammatory landscape, beyond a latent T. gondii infection elicited type 1-like neuroinflammation, display elevated production of IL-1α and IL-1β in the hippocampus. Using a mouse model to specifically delete the IL-1 receptor from excitatory neurons, they uncovered that neuronal IL-1 signaling is required for the spatial memory deficits caused by latent T. gondii infection. By singling out a chronic exposure to IL- ip they found that IL-1β signaling in neurons indeed impairs spatial memory. Moreover, they showed that chronic exposure to IL-1β increases DNA DSB levels in neurons, and that neuronal H2A.X-dependent DSB response mediates the IL-1β-induced memory deficits. Importantly, neuronal DSBs were also increased upon T. gondii infection and the deficit in hippocampus- dependent spatial memory caused by T. gondii infection was prevented by the abrogation of neuronal H2A.Xdependent signaling. These results highlight the instrumental role of cytokine- induced DSB-dependent signaling in spatial memory defects elicited by a chronic infectious neuroinflammatory process. Accordingly, the present invention relates to a method of treating a subject suffering from cognitive deficit comprising administering to said subject a therapeutically effective amount of an inhibitor of IL- 1 -induced H2A.X signaling.

Description

METHODS OF TREATING COGNITIVE DEFICIT

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular neurology.

BACKGROUND OF THE INVENTION:

Chronic neurological diseases are devastating pathologies with a major medical and economic burden. Although their underlying etiologies and molecular mechanisms are not fully understood, the neuronal inflammatory response has been identified as a key player in neurological dysfunction. Chronic neuroinflammation may be sterile or caused by the invasion and persistence of microorganisms in the central nervous system (CNS), such as viruses or parasites¹. Although such infections may seem mostly asymptomatic, the persistence of a pathogen in the CNS substantially shapes the local innate and adaptive immune responses, with potential long-term consequences on brain function such as inflammaging and even neurodegeneration²'⁴. For pathogens that establish persistent infections in the CNS, including Herpesviruses, Bornavirus, and Toxoplasma gondii (T. gondii), intricate relationships between host neural cells, immune responses and the pathogens are determinant for disease outcome. T. gondii is an intracellular apicomplexan parasite that can establish long-lasting CNS infection in all warm-blooded animals, including humans. Thirty (30) to 50% of the worldwide population is estimated to display a positive serology for (i.e. has been exposed to) T. gondii⁵. Although this infection can lead to a deadly encephalitis in immunocompromised patients due to parasite reactivation in the brain, most cases of infection are seemingly clinically silent thanks to an effective immune control of the parasite during the acute and chronic stages, and hence are called "latent". Nonetheless, a substantial body of evidence suggests that latent T. gondii infection contributes to several human neuropsychiatric disorders such as schizophrenia, bipolar disorder, obsessive compulsive disorder and epilepsy⁶'¹⁰. Studies of T. gondii experimental infection in the mouse, one of the parasite’s natural hosts, have indicated that the brain parasite burden, the level of neuroinflammation, and the extent of neurological symptoms depend on the combination of parasite strain and mouse genetic background, most likely since these processes are influenced by the efficiency of parasite immune control in the brain. Several (neuro)immune cells contribute to T. gondii immune surveillance in the CNS, including microglia¹¹, astrocytes^{12 13}, monocytes¹⁴, and CD8+ T lymphocytes, which play a central role^{15 16}. It follows that C57BL/6 mice are naturally susceptible to develop encephalitis upon infection by type II T. gondii. However, infection of C57BL/6 mice with type II parasites which are genetically engineered to express a model antigen that is efficiently processed and presented by major histocompatibility class I (MHCI) molecules, elicits protective CD8+ T cell responses providing effective brain parasite control, and allowing the establishment of latency¹⁷. So far, most behavioral and cognitive impairments have been studied in the context of T.gondii-induced chronic encephalitis. Exploration of the contribution of latent infection to behavioral alterations remains limited to innate and anxiety-related behavior, whereas the impact on more complex cognitive functions (e.g. memory) remains ill-defined^{18 19}. Therefore, studies are needed to understand the neural processes that may be impacted by latent T. gondii infection, and may cause cognitive dysfunction. Interestingly, recent evidence have suggested a detrimental role for neuroinflammation in T.gondii-induced behavioral alterations¹⁹'²¹. However, a full understanding of the causes of neuronal dysfunction upon infection is challenging, due to the intricate intervention of inflammatory and neuronal components in the pathology. The ability of the immune response to control infection relies in great part on the production of cytokines. Cytokines are known to play both beneficial and detrimental roles in the CNS²²'²⁶. While low levels of Interferon-g²⁷, Interleukins (IL)-17²⁸, IL-4²⁹, IL-la³⁰, IL-33³¹ play important neuromodulatory roles underlying social behavior, short-term or fear memory encoding, respectively, IL-iβ, IL6, IL-17, IL-33³²’³³, Tumor Necrosis Factor-a, and Interferon a and y also mediate chronic pathological signaling, which in turn leads to neuronal dysfunction^22,34. Chronic elevated circulating IL-1, IL-6, TNF-α , or IFN-a leads to persistent alterations in neurotransmitters like glutamate, and impairs neuronal growth factor function²⁵’³⁵. Thus, although the molecular mechanisms underlying the conversion of cytokine signals into neuro-pathological factors have been partly described²², the mechanisms by which they exert a direct and long-lasting impact on neuronal function require further investigation. One hypothesis to explain the long-lasting nature of behavioral alterations, is that inflammation could perturb epigenetic mechanisms. Notably, indirect evidence suggests that pro- inflammatory cytokines modulate the epigenome of neural cells³⁶'³⁸. Epigenetics is considered as a key regulator of neuronal function, because of its highly dynamic action that may durably impact gene expression. DNA double strand breaks (DSB) are now considered as a central regulator of neuronal epigenetics involved in cognition³⁹'⁴¹. In neurodegenerative conditions such as Alzheimer's disease (AD), it was shown that DSB and their marker, phosphorylated histone variant H2A.X, accumulate in neurons, because of impaired repair mechanisms due to disturbed electrical activity of neuronal networks in the brain, leading to cognitive decline^40,41. Interestingly, similar alterations in electrical activity are also detected during exposure to pro- inflammatory cytokines^22,42,43.

SUMMARY OF THE INVENTION:

The invention is defined by the claims. In particular, the present invention relates to a method of treating a subject suffering from cognitive deficit comprising administering to said subject a therapeutically effective amount of an inhibitor of IL- 1 -induced H2A.X signaling.

DETAILED DESCRIPTION OF THE INVENTION:

Here, the Inventors show that chronic IL- 1 -induced H2A.X signaling, which mediated DNA DSB response, in hippocampal neurons is a critical driver of cognitive deficits in chronic low grade neuroinflammation. They show that mice chronically infected with the brainpersisting T. gondii parasite display deficits in spatial memory consolidation without general memory loss. Extensive mapping of the neuroinflammatory landscape, beyond a latent T. gondii infection elicited type 1-like neuroinflammation, display elevated production of IL-la and IL-ip in the hippocampus. Using a mouse model to specifically delete the IL-1 receptor from excitatory neurons in adult mice, they uncovered that neuronal IL-1 signaling is required for the spatial memory deficits caused by latent T. gondii infection. By singling out a chronic exposure to IL-ip they found that IL-ip signaling in neurons indeed impairs spatial memory. Moreover, they showed that chronic exposure to IL-ip increases DNA DSB levels in neurons, and that neuronal H2A.X-dependent DSB response mediates the IL-ip-induced memory deficits. Importantly, neuronal DSBs were also increased upon T. gondii infection and the deficit in hippocampus-dependent spatial memory caused by T. gondii infection was prevented by the abrogation of neuronal H2A.X-dependant signaling.

In a first aspect, the present invention relates to a method of treating a subject suffering from cognitive deficit comprising administering to said subject a therapeutically effective amount of an inhibitor of IL- 1 -induced H2A.X signaling. In some embodiments, the inhibitor of IL-1- induced H2A.X signaling is selected from the list consisting in an IL-1 inhibitor, an IL-1R inhibitor or an H2AX inhibitor As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine or a primate. In a preferred embodiment, the subject is a human. In some embodiments, the subject is an adult aged forty-five or over. In some embodiments, the subject is an elderly subject. As used herein the term “elderly subject” refers to an adult aged sixty or over. In some embodiments, the subject suffers from attention, learning, intelligence, language, memory, spatial memory, precision memory, perception, judgement, mental acuity, decision making, problem solving or reasoning trouble. In some embodiments, the subject is at risk of suffering from cognitive deficit. In some embodiments, the subject suffers from a neuroinflammation. In some embodiments, the subject suffers from chronic neuroinflammation. In some embodiments, the subject suffers from encephalitis. In some embodiments, the subject suffers from a neurodegenerative condition (e.g. dementia, Alzheimer’s disease, Parkinson’s disease). In some embodiments, the subject suffers from Alzheimer’s disease. In some embodiments, the subject suffers from a neurological disease (e.g. depression, brain injury, bipolar disorders, schizophrenia, obsessive compulsive disorder, epilepsy). In some embodiments, the subject suffers from a neuroinfection (e.g. Herpesvirus, Bornavirus, Toxoplasma Gondii). In some embodiments, the subject suffers from acute or chronic Toxoplasma Gondii infection.

As used herein, the term “cognition” has its general meaning in the art and refers to a set of mental processes involved in brain functions related to knowledge (e.g. attention, learning, intelligence, language, memory, spatial memory, precision memory, perception, judgement, mental acuity, decision making, problem solving, reasoning...).

As used herein, the term “cognitive deficits” refers to an impairment in one or more cognitive abilities. In order to measure cognitive deficits, numerous clinical tests are available. As example, the Mini-Mental State Examination (MMSE®) or "Folstein test” is a test of cognitive evaluation and memory capacities which helps to detect the presence of dementia or to monitor cognitive evolution (« Mini-Mental State: A Practical Method for Grading the Cognitive State of Patients for the Clinician », Folstein M. et al., Journal of Psychiatric Research, 1975, vol. 12, n° 3, pp. 189-198). Other examples include the “Montreal Cognition Assessment” or “MOCA®”, the “General Practitioner Cognition” or “GP-COG”, the “6-item impairment test” or “6-CIT” or the “clock-drawing test”. As used herein, the terms “treating”, “treatment” or “therapy” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. In some embodiments, the term “treatment” particularly refers to the preventive treatment of cognitive deficits. The treatment may be administered to a subject having a medical disorder or a subject likely to suffer from the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. IL-1, IL-1R or H2AX inhibitor) into the subject, such as by intrathecal, intraocular, mucosal, intradermal, intravenous, intranasal, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

As used herein, the term “efficient” denotes a state wherein the administration of one or more drugs to a subject permit to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or to prolong the survival of a subject beyond that expected in the absence of such treatment. A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

As used herein, the term “IL-1” or “Interleukin-1” refers to a protein member of the interleukin 1 cytokine family. The term encompasses IL-la, IL-ip, IL-IRA, IL-18, IL-36Ra, IL-36a, IL- 37, IL-36P, IL-36y, 11-38 and IL-33. In some embodiments, the IL-1 cytokine is IL-la. In some embodiments, the IL-1 cytokine is IL-ip.

As used herein, the term “IL-la” refers to a protein member of the interleukin 1 cytokine family (NCBI Gene : 3552; Ensembl: ENSG00000115008).

An exemplary amino acid sequence of IL-la is depicted as SEQ ID NO:1 : SEQ ID NO : 1 >sp | P01583 | ILIA HUMAN Interleukin- 1 alpha OS=Homo sapiens OX=9606 GN=IL1A PE=1 SV=1

MAKVPDMFED LKNCYSENEE DSSSIDHLSL NQKSFYHVSY GPLHEGCMDQ SVSLSI SETS KTSKLTFKES MVWATNGKV LKKRRLSLSQ SITDDDLEAI ANDSEEEI IK PRSAPFSFLS NVKYNFMRI I KYEFILNDAL NQSI IRANDQ YLTAAALHNL DEAVKFDMGA YKSSKDDAKI TVILRI SKTQ LYVTAQDEDQ PVLLKEMPEI PKTITGSETN LLFFWETHGT KNYFTSVAHP NLFIATKQDY WVCLAGGPPS ITDFQILENQ A

As used herein, the term “IL-ip” refers to a protein member of the interleukin 1 cytokine family (NCBI Gene : 3553; Ensembl: ENSG00000125538).

An exemplary amino acid sequence of IL-ip is depicted as SEQ ID NO:2 :

SEQ ID NO : 2 >sp | P01584 | IL1B HUMAN Interleukin- 1 beta OS=Homo sapiens OX=9606 GN=IL1B PE=1 SV=2

MAEVPELASE MMAYYSGNED DLFFEADGPK QMKCSFQDLD LCPLDGGIQL RI SDHHYSKG FRQAASVWA MDKLRKMLVP CPQTFQENDL STFFPFI FEE EPI FFDTWDN EAYVHDAPVR SLNCTLRDSQ QKSLVMSGPY ELKALHLQGQ DMEQQWFSM SFVQGEESND KI PVALGLKE KNLYLSCVLK DDKPTLQLES VDPKNYPKKK MEKRFVFNKI EINNKLEFES AQFPNWYI ST SQAENMPVFL GGTKGGQDIT DFTMQFVSS

As used herein, the term “IL-1R” or “Interleukin-1 Receptor” refers to a cytokine receptor that belongs to the interleukin- 1 receptor family. In some embodiments, the IL-1 receptor is IL-1RI (NCBI Gene : 3554; Ensembl: ENSG00000115594).

An exemplary amino acid sequence of IL-1R1 is depicted as SEQ ID NO:3 :

SEQ ID NO : 3 >sp | P14778 | IL1R1 HUMAN Interleukin- 1 receptor type 1 OS=Homo sapiens OX=9606 GN=IL1R1 PE=1 SV=1 MKVLLRLICF IALLI SSLEA DKCKEREEKI ILVSSANEID VRPCPLNPNE HKGTITWYKD DSKTPVSTEQ ASRIHQHKEK LWFVPAKVED SGHYYCWRN SSYCLRIKI S AKFVENEPNL CYNAQAI FKQ KLPVAGDGGL VCPYMEFFKN ENNELPKLQW YKDCKPLLLD NIHFSGVKDR LIVMNVAEKH RGNYTCHASY TYLGKQYPIT RVIEFITLEE NKPTRPVIVS PANETMEVDL GSQIQLICNV TGQLSDIAYW KWNGSVIDED DPVLGEDYYS VENPANKRRS TLITVLNI SE IESRFYKHPF TCFAKNTHGI DAAYIQLIYP VTNFQKHMIG ICVTLTVI IV CSVFIYKI FK IDIVLWYRDS CYDFLPIKAS DGKTYDAYIL YPKTVGEGST SDCDI FVFKV LPEVLEKQCG YKLFIYGRDD YVGEDIVEVI NENVKKSRRL I I ILVRETSG FSWLGGSSEE QIAMYNALVQ DGIKWLLEL EKIQDYEKMP ESIKFIKQKH GAIRWSGDFT QGPQSAKTRF WKNVRYHMPV QRRSPSSKHQ LLSPATKEKL QREAHVPLG

As used herein, the term “H2AX” refers to a protein encoded by H2AFX gene (NCBI gene: 3014; Ensembl: ENSG00000188486).

An exemplary amino acid sequence of H2AX is depicted as SEQ ID NO:4 : Histone H2AX OS=Homo sapiens OX=9606

MSGRGKTGGK ARAKAKSRSS RAGLQFPVGR VHRLLRKGHY AERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRI I PRHLQLAIRN DEELNKLLGG VTIAQGGVLP NIQAVLLPKK TSATVGPKAP SGGKKATQAS QEY Those skilled in the art will understand that the sequences may be subject to species-related variation of sequences.

As used herein, the term “IL-1 inhibitor”, “IL-1R inhibitor” or “H2AX inhibitor” denotes a molecule that partially or totally inhibits the biological activity or expression of IL-1, IL-1R or H2AX. In some embodiments, the IL-1R inhibitor is an antagonist of IL-1R. In some embodiments, the inhibitor of IL- 1 -induced H2A.X signaling impairs the interaction between IL-1 and IL-1R. In some embodiments, the IL-1, IL-1R or H2AX inhibitor interacts directly with IL-1, IL-1R or H2AX. In some embodiments, the IL-1, IL-1R or H2AX inhibitor impairs neuronal DNA double-strand break by inhibiting IL- 1 -induced H2A.X signaling. In some embodiments, the IL-1 inhibitor is an IL-la inhibitor. In some embodiments, the IL-1 inhibitor is an IL-ip inhibitor.

In some embodiments, the IL-1, IL-1R or H2AX inhibitor according to the invention is an inhibitor of IL-1, IL-1R or H2AX gene expression. In some embodiments, the IL-1, IL-1R or H2AX inhibitor is an inhibitor of IL-1, IL-1R or H2AX gene expression selected from the list consisting of antisense oligonucleotide, nuclease, siRNA, shRNA or ribozyme. In some embodiments, the inhibitor of IL-1, IL-1R or H2AX gene expression is a siRNA directed against IL-1, IL-1R or H2AX gene. In some embodiments, the inhibitor of IL-1, IL-1R or H2AX gene expression is a shRNA directed against IL-1, IL-1R or H2AX gene. Thus, in some embodiments, the inhibitor of IL-1, IL-1R or H2AX gene expression is a siRNA or a shRNA directed against IL-1, IL-1R or H2AX gene. IL-1, IL-1R or H2AX gene expression can be reduced by contacting a subject or cell with a small single or double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that IL- 1, IL-1R or H2AX gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

In some embodiments, the IL-1, IL-1R or H2AX inhibitor is an antisense oligonucleotide. As used herein, the term "antisense oligonucleotide (AON)" refers to an oligonucleotide capable of interacting with and/or hybridizing to a pre-mRNA or an mRNA having a complementary nucleotide sequence thereby modifying gene expression. Typically, the antisense oligonucleotide is complementary to the nucleic acid sequence that is necessary for preventing splicing of the targeted exon including cryptic exon, supplementary exon, pseudo-exon or intron sequence retained after splicing. In a more particular embodiment, the IL-1, IL-1R or H2AX inhibitor is an antisense oligonucleotide directed against IL-1, IL-1R or H2AX. In some embodiments, the antisense oligonucleotide is an IL-1, IL-1R or H2AX antisense oligonucleotide.

Ribozymes can also function as inhibitors of IL-1, IL-1R or H2AX gene expression in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of IL-1, IL-1R or H2AX mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of IL-1, IL-1R or H2AX gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, antisense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and, in particular, to the cells expressing IL-1, IL-1R or H2AX. In the scope of the present invention, the vector is particularly able to facilitate the transfer of the oligonucleotide siRNA or ribozyme nucleic acid to neurons. Particularly, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a Bornavirus. One can readily employ other vectors not named but known to the art. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. In a preferred embodiment, the viral vector is a lentivirus. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high- efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991. Preferred viruses for certain applications are the adenoviruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wildtype adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of intrathecal, parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation. In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters. In some embodiments, an endonuclease can be used to abolish the expression of IL-1, IL-1R or H2AX. As an alternative to, as example, cDNA overexpression or downregulation by RNA interference, more recent technologies provide means to manipulate the genome. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiments, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e267L), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics. H3.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.1 L), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci. In some embodiments, the endonuclease is CRISPR-Cpfl which is CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

In some embodiments, the IL-1, IL-1R or H2AX inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not). The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

In some embodiments, the IL-1, IL-1R or H2AX inhibitor is a PROTAC. The term PROTAC or PROteolysis TArgeting Chimera refers to a heterobifunctional molecule composed of two active domains and a linker, capable of removing specific unwanted proteins by inducing selective intracellular proteolysis. PROTACs are typically constituted of two covalently linked protein-binding molecules, the first one capable of engaging an E3 ubiquitin ligase and the second one that binds to a target protein meant for degradation. The recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein via the proteasome. PROTAC strategies are described as example in Schneekloth A.R. et al. (Ashley R. Schneekloth et al. “Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics” Bioorganic & Medicinal Chemistry Letters, Volume 18, Issue 22, 2008, Pages 5904-5908), Jia X. et al. (Xiaojuan Jia et am. “Targeting androgen receptor degradation with PROTACs from bench to bedside”, Biomedicine & Pharmacotherapy, Volume 158, 2023, 114112) or Sakamoto KM et al. (Sakamoto K.M. et al. “Protacs: chimeric molecules that target proteins to the Skpl-Cullin-F box complex for ubiquitination and degradation”. Proc Natl Acad Sci U S A. 2001;98(15):8554-8559. doi: 10.1073/pnas.141230798).

In some embodiment, the IL-1, IL-1R or H2AX inhibitor according to the invention is an antibody directed against IL-1, IL-1R or H2AX. This embodiment includes, as example, canakinumab (CAS N°914613-48-2) or gevokizumab (CAS N°1129435-60-4). Antibodies directed against IL-1, IL-1R or H2AX can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against IL-1, IL-1R or H2AX can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-IL-1, anti -IL- 1R or anti-H2AX single chain antibodies. Compounds useful in practicing the present invention also include anti -IL- 1, anti-IL-lR or anti-H2AX antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to IL-1, IL-1R or H2AX. Humanized anti-IL-1, anti-IL-lR or anti-H2AX antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397). Then, for this invention, neutralizing antibodies of IL-1, IL-1R or H2AX are selected.

In another embodiment, the antibody according to the invention is a single domain antibody directed against IL-1, IL-1R or H2AX. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals, which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences, which define the binding affinity and specificity of the VHH. The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation. VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B- cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In some embodiments, the inhibitor according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

In some embodiments, the IL-1, IL-1R or H2AX inhibitor is a polypeptide. This embodiment includes, as example, anakinra (CAS N° 143090-92-0), rilonacept (CAS N°501081-76-l). In one embodiment, the polypeptide of the invention may be linked to a “cell-penetrating peptide” to allow the penetration of the polypeptide in the cell. The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012). The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Particularly, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is particularly generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant proteins, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli. In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters. A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long- circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa). In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Toxoplasma gondii chronic infection impairs the precision and consolidation of spatial memory in a severity-dependent manner. Starting 12 weeks post-infection by Tg.GRA6-OVA (latency) vs. Tg.SAGl-OVA (encephalitis) parasites, or PBS (uninfected), C57BL6/J mice were tested in a series of behavioral tests: the Barnes maze (A-F), the novel object recognition test (G-H) and the object location tasks (I- J). A-B) In the Barnes maze, learning curves show mean daily distance run (A), or mean numbers of errors of holes visits (B) prior to find the target hole connected to a hidden exit box. Effect of the time (p<0.0001) and Tg strain (p<0.001) with no interaction as analyzed by repeated measures mixed-effects models, and Dunnett pos-hoc tests compared to the non-infected group. (C-D) The strategy used by mice to find the target holes was determined. C) Representative trajectory heatmaps illustrating the four types of strategies used to find the target hole indicated by the white arrow. White dashed lines delineate quadrants. Al : adjacent quadrant 1, T: target quadrant, A2: adjacent quadrant 2, Op: opposite quadrant. D) Mean distribution of daily strategies in the course of the 6 days of training and the probe trial as shown as percentage per group. Freeman- Halton extension of the Fisher 's exact test using 3 groups (mixed and spatial groups were merged) was used to compare distributions with uninfected mice. E) Percent of time, mice spent in the target quadrant vs. the three other quadrants during a 90-s probe trial run 24 h after the last training trial in the Barnes. Results were tested by the One-sample t-test compared to chance (25%). F) Number of visits of the original exit hole (Target) location vs. each other hole location in other quadrants (Other) during the probe trial. Results were analyzed by Dunnett post-hoc tests compared to the noninfected group or by paired t-test as indicated by brackets, n = 11-16 mice per group of treatment from 2 independent cohorts. (G-H) General long-term memory assessed in a novel object recognition task, 24 h after training. Bars in H) represent the time mice spent exploring the object that is novel (New, N) or not (Old, O), expressed as percentage of the total time spent exploring either object (shown in G). (I- J) Consolidation of spatial memory was assessed in the object location task, 3 h after training. Bars in J) represent the time mice spent exploring the object that has been moved (M) or not (unmoved, U), expressed as percentage of the total time spent exploring either object (shown in I). (G-J) n = 10-20 mice per group of treatment from 2 independent cohorts. Results were tested in G-J by One-sample t-test compared to chance (50%). ns, non-significant, *p<0.05, **p<0.01, ***p<0.001. Values are means ± s.e.m.

Figure 2. Neuronal IL-l-dependent signaling pathways drive cognitive impairment caused by Tg chronic infection. (A-F) Consolidation of spatial memory was assessed in the object location task 3 h after training of Zg-infected vs. non infected transgenic mice, for which the IL-1R1 receptor can be knocked out in glutamatergic excitatory neurons of the forebrain (IllrlneuronKO) upon induction of the recombination by Tamoxifen treatment. (A) IL-ip concentration in mouse hippocampus at 7 wpi (Kruskal -Wallis, Dunn’s multiple comparison test, n = 4-13 mice per group from one experiment). (B-C) Object location task: Bars in C) represent the time mice spent exploring the object that has been (M) or not (unmoved, U), expressed as percentages of the total time spent exploring either object (shown in B). n = 1 1 - 14 mice per group of treatment from 2 independent cohorts (one-sample t test compared to chance at 50 %. (D) Percentage of initial body mass throughout infection n = 4-13 mice per group from one experiment (Kruskal-Wallis and Dunn’s multiple comparison test performed on the area under the curve). (E) Cyst number and (F) total parasite burden in the brain n= 11- 13 infected mice per group from one experiment (unpaired Student's t test), ns, nonsignificant, *p<0.05, **p<0.01, ***p<0.001. Values are means ± s.e.m.

Figure 3. Chronic exposure to IL-ip induces deficits in the consolidation and the precision of spatial memory. C57BL/6J male mice were subcutaneously implanted with osmotic minipumps containing saline or IL-ip diluted in saline solution (5 pg/kg/day) for 28 days or 35 days. Starting 3 weeks post-implantation, mice were tested in a series of behavioral tests: the Barnes maze (B-F), the novel object recognition test (G) and the object location tasks (H-I). A) IL-ip concentrations in the serum were assessed by ELISA in Saline (saline + carrier) controls or IL- ip group of mice. n= 11-16 mice per group from two independent experiments (unpaired Student's t test). (B-F) In the Barnes maze, learning curves show mean daily distance run (B), or mean numbers of errors of holes visits (C) prior to find the target hole connected to a hidden exit box. Effect of the time (p<0.0001) was analyzed by repeated measures mixed-effects models. D) Mean distribution of daily strategies used by mice to find the target holes in the course of the 6 days of training and the probe trial are shown as percentages per group. Freeman- Halton extension of the Fisher’s exact test using 3 groups (mixed and spatial groups were merged) was used to compare distributions. E) Percent of time, mice spent in the target quadrant vs. the three other quadrants during a 90-s probe trial run 24 h after the last training trial in the Barnes. Results were tested by the One-sample ttest compared to chance (25%). F) Number of visits of the original exit hole (Target, T) location vs. each other hole location in other quadrants (Other) during the probe trial. Results were analyzed by Student-t tests compared to the Saline group or by paired Etests between T and O as indicated by brackets, n ------ 14-17 mice per group of treatment from 2 independent cohorts. G) The novel object recognition task was performed 24 h after a training step with two identical obj ects. Bars represent the time mice spent exploring the object that is novel (New) or not (Old), expressed as percentage of the total time spent exploring either object. (H-I) The object location task was performed 3 h after training on wildtype non transgenic mice (H, n = 13-14 mice per group of treatment from 2 independent cohorts) and on Illrl^neuronKO versus II Ir 1^WT mice (I, n = 8-14 mice per group of treatment from 2 independent cohorts) 24 days postimplantation. Bars represent the time mice spent exploring the object that has been (M) or not (unmoved, U), expressed as percentages of the total time spent exploring either. Results were tested by One-sample t-test compared to chance (50%). ns, non-significant, *p<0.05, **p<0.01, ***p<0.001. Values are means ± s.e.m.

Figure 4. Chronic Toxoplasma gondii infection or exposure to IL-ip increase neuronal levels of DNA double-strand breaks. (A-C) DG cells and neurons in the CAI-3 with 53BP1- positive were counted and averaged on three coronal sections per mouse, upon chronic latent Tg (A, n = 4-9 mice per condition), encephalitic Tg infection (B, n= 3-4 mice per condition) or at 28 days post-implantation of IL-ip- versus saline-infusing minipumps (C, n = 18-19 mice per condition). D) Cultures of primary hippocampal neurons from WT mice were exposed to the indicated concentrations of mouse IL- lb (+) or vehicle (0) for 5 h. Levels of the DSB marker yH2A.X were determined by western blotting. The average yH2A.X signals to a-tubulin ratio in vehicle-treated cultures was defined as 1.0. n = 7-23 wells per condition from 11 independent experiments. In western blots, each lane contained a sample from a different culture well, ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001 vs. uninfected (A, B) or saline (C), or vehicle (D) by Kruskal-Wallis and Dunn’s multiple comparison post-hoc tests. Bars represent means ± s.e.m.

Figure 5. Knocking out H2A.X in excitatory neurons is sufficient to prevent the deleterious effects of chronic Toxoplasma gondii infection and exposure to IL-ip on memory consolidation. (A-B) The object location task was performed 3 h after training with H2A.X^WT and H2A.X^neuronKO, 10 weeks after Tamoxifen treatment, and either 24 days postimplantation of IL- Ip- versus saline (Sal.)-infusing mini pumps A), n = 9-11 mice per condition from 2 independent cohorts) or 5 weeks post-infection by encephalitic Tg strain (Tg, B, n ------ 7- 10 mice per condition from 2 independent cohorts). C) Cyst number and total parasite burden in the brain was assessed from one experiment (n = 5 mice per condition). Bars represent the time mice spent exploring the object that has been moved, expressed as percentages of the total time spent exploring either of the two objects. Results were tested by One-sample t-test compared to chance (50%) (A, B) or Student's t-test (C). ns, non-significant, *p<0.05,

**p<0.01. Values are means ± s.e.m. EXAMPLE:

Material and Methods

Mice, infection and tissues sampling. Specific pathogen-free C57BL/6J male mice were purchased from Janvier Laboratories (France) at 6-8 weeks of age. Mice were acclimated for 1-2 weeks in the experimental BSL2 facility. We also studied CaMKIIa-CRE-ERT2+/-: II- transgenic male mice. CaMKIIa-CRE-

ERT2-/-(Cre negative) littermates of each strain were use as controls. All mice were on a pure C57BL6/J background⁴⁴'⁴⁶. Transgenic mice were bred at the UMS006-ANEXPLO-CREFRE facility (Toulouse). To induce the knockout of the floxed gene, 6-8 week-old transgenic mice were injected daily intraperitoneally (ip) by a suspension of Tamoxifen (Sigma-Aldrich) in corn oil (Sigma-Aldrich) for 5 days at 10 mg kg-1 day-1. Commercial and transgenic mice used in this study were implanted with osmotic minipumps (Alzet AZT2004, Charles River) at 8 weeks of age or were injected intraperitoneally (i.p.) with tachyzoites or mock injected with PBS (uninfected group) between 9 and 16-weeks of age. Behavior assessments were carried out starting 24 days post-implantation or between 6 and 16 weeks post-infection. Mice were acclimated for one week in the experimental ABSL2 facility, prior to intraperitoneal (i.p.) injection with 200 Pru- or 2000 ME49-derivatives tachyzoites in 200 pL of PBS, or with 200 pL of PBS only for the uninfected group. During acute infection, mice were observed daily and dietary supplement (DietGel® Recovery, Clear H2O) was added in all experimental conditions. Mice were routinely weighed throughout infection. Littermates were group-housed. For testing in the Barnes maze, mice were single-housed from 3 days before the start of training until the test was concluded. All mice were housed in cages with enrichment (nesting material and shelter) at 24°C, had ad libitum access to irradiated A4 food (R04-10, SAFE Diets) and water and were exposed to a 12-h light/dark cycle. For histological and biochemical analyses, mice were anesthetized with Avertin (tribromoethanol, 250 mg kg-1, ip) for IL-lb infusion experiments or with a mix of ketamine (200 mg kg-1) and xylazine (20 mg kg-1) injected i.p for parasite-related experiments and assessments of the immune response. Mice were then perfused transcardially with 0.9% NaCl. One hemibrain was used fresh for parasite load analysis or flow cytometry, or snap frozen and stored at -80°C for bulk RNA sequencing, cytokine/chemokine multiplex immunoassay or biobanking. The other hemibrain was drop- fixed in 4% paraformaldehyde in phosphate-buff ered saline (PBS) and sectioned (30 pm) with a sliding microtome (Leica SM2000R). All mouse experiments were performed in accordance with European Union Council Directive 86/609ZEEC, and experiments were performed following the French national chart for ethics of animal experiments (articles R 214-87 to -90 of the “Code rural”). Our protocols received approvals from the local committee on the ethics of animal.

Tg parasite strains and culture. Derivatives of type II Tg strains (Prugniaud and ME49) were used in this study (see Table 1 for details). For both strains, Tg tachyzoites were maintained in vitro by serial passage on confluent monolayer of human foreskin fibroblasts (HFF, ATCC SCRC-1041) in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 1% (vol/vol) fetal bovine serum (FBS, Gibco), 1% (vol/vol) penicillin/ streptomycin (Gibco), 0,1% (vol/vol) 2-mercaptoethanol (Gibco). For in vivo infections, parasites were prepared immediately before mouse infection. Infected HFF were scraped and remaining intracellular tachyzoites were released through homogenization with a 23G needle. Freshly egressed parasites were filtered through a 3 pm polycarbonate hydrophilic filter (it4ip S. A.), centrifuged at 1000 g for 10 min and resuspended in phosphate-buffered saline (PBS, Sigma- Aldrich). Tachyzoites were individualized through a 25G needle, counted and diluted to 103 or 104 parasites/ml.

Osmotic minipumps implantation. Mouse recombinant IL- lb (Immunotools GmbH, Germany) was dissolved in sterile saline solution 80 (0.9% sodium chloride) containing 0.1% Bovine serum albumin as a carrier. For the chronic experiments, mice were implanted s.c; in the interscapular region with osmotic minipumps (model AZT2004, Alzet, Charles River). Minipumps were filled with saline (saline + carrier) or IL- lb solutions per the manufacturer's instructions. Model 2004 minipumps delivered fluid at a rate of 0.25 pL/h for up to 35 days. Concentration of IL-lb in each minipump was calculated to infuse 5 pg. kg-1. day-1⁵⁰. After 24h of priming in saline solution at 37°C, mini pumps were surgically implanted in mice anesthetized with Avertin (tribromoethanol, 250 mg. kg-1, AlphaAesar, Merck). S.c. injections of 0.05 mg. kg-1 of Buprenorphine (AnimalCare) were use perioperatively to prevent pain during the recovery of the mice. 2 weeks post-implantation, to prevent minipumps explants, mice were lightly anesthetized with 4% Isoflurane, potential skin adherences at the tip of the pumps were disconnected and a surgical clip (Reflex clip 9 mm, WPI) was placed instead.

Behavioral testing. For all behavioral tests and subsequent analyses, experimenters were blinded to the genotype and treatment of mice. Mice were assessed in the following tests in the indicated sequence and as described in the corresponding references: elevated plus maze (EPM)⁵¹, object location (OL) and novel object recognition^51,52 and the Barnes maze, with the following modifications. In the EPM, videotracking and analysis were performed using ANYMaze software (Ugobasile, Italy). In the OL and the NOR, after an initial step of exploration of two identical objects in a large arena (60 x 30 cm) for 10 minutes, mice were allowed to explore again the arena 3 hours later for 10 minutes, when one of the familiar object had been moved (OL). 24h later, one familiar object was replaced by a novel object in terms of shape, texture and colors, and mice explored the arena for 10 minutes. Activity of the mice was videorecorded using camcorder and videos converted into mp4 format and subsequently analyzed: i) automatically by a homemade software (coded with Python) to exclude any side preference bias and record total distance ran ; ii) manually analyzed using a free video annotation tool (ANVIL, Germany) to determine times and pokes of exploration of the objects. A mouse was considered as exploring an object when its nose was in contact with the object and its four paws were on the ground. In the Barnes maze, a cued-box trial preceded the hidden- exit box trainings, consisting in a first trial of releasing the mouse in the middle of the maze for 3 minutes, with an exit box linked to the target hole, hidden under the table of the maze with a reward cereal loop inside it. Passed the three minutes of each trial, if the mouse had not found the exit box, it was guided by the experimenter to it and received a cereal loop as a reward as soon as it had found the box. 4 hidden-exit box trainings trials per day for Tg related experiments and 3 trials per day for pumps-related experiments were performed during 5 days to allow each control groups to reach a steady plateau in their performances without overtraining. Cohorts of mice implanted with osmotic minipumps undergo either EPM, OL and NOR or EPM and Barnes maze.

Immunocytochemistry. Immunostaining of mouse brain sections was performed essentially as described (Suberbielle 2015). TBS was used and steps were added for antigen retrieval and peroxidase inhibition to enhance nuclear staining. Blocking solution was 5% normal goat serum in 0.1% TBS-Tween20. Sections were incubated overnight at 4 °C with primary antibodies, including monoclonal mouse anti-GFAP (clone GA5, Millipore); polyclonal chicken anti-GFP and rabbit anti-53BPl (Novus Biologicals), rabbit anti-doublecortin (Synaptic systems), rabbit anti-IBA-1 (Wako) and chicken anti-MAP2 (ABCam). Primary antibodies were diluted 1 :500 (or 1: 1000 for anti-doublecortin) in blocking solution. Secondary antibodies were goat antimouse ot anti-rabbit Alexa 488, anti-rabbit Alexa 546 or Alexa 633 (Life technologies). Biotinylated anti-rabbit antibodies (Vector Laboratories) were used for doublecortin. Diaminobenzidine (Vector Laboratories DAB kit) was used as a chromogen. After extensive rinses, coverslips or sections were mounted with Prolong gold mounting medium (Life technologies). For quantification of GFAP-positive or IBA-1 -positive signals on DAPI, digitized images of dentate gyrus region were obtained with a digital brightfield Olympus BX41 microscope with a DPI 1 digital camera system and a X-citel20 Q (Lumen Dynamics) lamp or with Zen software (Zeiss) on a Zeiss Axio-observer widefield microscope. Image were manually annotated with cell counter tools from Image J, over three frames per mouse. Counts were converted into densities using the area covered by the counting in mm3. Tiles of z-stacks of images of 53BP1 foci or GFP and IBA-1 and GFAP signals were obtained with a Leica SP8 confocal microscope, at a 20X objective. Images were acquired and processed with LSM software. Z-stacks of merged mosaic confocal images were processed with Image J, and final images were obtained by orthogonal projection. 53BP1, and doublecortin staining in the DG from coronal sections of mice was quantified essentially as described⁵³. Counts of neurons positive with 53BP1 foci were averaged from 3 sections per mouse unless stated otherwise. Counts of doublecortin-positive neurons were averaged from 2-3 sections per mouse.

Cell cultures and treatments. Primary cultures of hippocampal neurons were established as described^52,53, using postnatal day 0 pups from pure C57BL6/J WT mice. Neurons were plated on 12-well plates (Coming) at 0.5 x 106 cells/well or on 12-mm glass coverslips coated with poly-D lysine (Millipore). Cultures were used for experiments at 14 DIV. Experimenters were blinded to treatments. Recombinant IL- lb (Immunotools GmbH, Germany) was dissolved at a stock concentration of lOOpg/ml in sterile Phosphate Buffer Saline containing 0.1% Bovine serum albumin as a carrier. Various concentrations were applied to neurons for 5 hours as described⁵³.

Western blot analysis. Cultured cells were washed in PBS and scraped into RIPA buffer according to⁵⁴. Briefly, lysates were sonicated for 10 min at 4°C and spun in a refrigerated microcentrifuge at maximal speed for 10 min. Protein concentrations were determined by Bradford assay. Proteins (20 pg per mouse sample) were loaded on 4-12% Bis-Tris gels (Life technologies), separated by SDS-PAGE, and transferred to nitrocellulose membranes. After Ih in blocking solution (5% nonfat milk in Tris-buffered saline (TBS)), membranes were incubated with primary antibodies in 5% nonfat milk in TBS/0.1% Tween for 3 h at room temperature for monoclonal dilution 1/1,000 (ThermoFisher, MAI-2022) and mouse anti-a-Tubulin monoclonal, dilution 1/10,000 (Sigma, T6199) ; Blots were washed 3 times in TBS/0.1% Tween and incubated with secondary IRD-tagged antibodies (CF770, Biotium) diluted 1 : 10,000 in Odyssey blocking buffer for 1 h at room temperature. Western blot signals were analyzed using an Odyssey Li-COR infrared imaging system coupled with Image studio software (Li-COR). All protein signals were normalized on a-Tubulin signal.

Hippocampus isolation and cell preparation for flow cytometry analyses. Hippocampus regions of the brain were microdissected and collected in cold Hanks’ Balanced Salt Solution (HBSS, Sigma- Aldrich), on ice-cooled plate under dissecting binocular. Hippocampus was then sliced with a scalpel and digested for 1 h at room temperature in HBSS supplemented with 0,5 mg/ml collagenase D (Roche) and 10 pg/ml DNase I (Sigma- Aldrich). During enzymatic digestion, tissue was mechanically dissociated by trituration with plastic pipette every ten minutes. Digestion was stopped by adding 10% (vol/vol) FBS in digestion mix, then samples were filtered through a 70 pm strainer (Falcon), washed with HBSS supplemented with 5% (vol/vol) FBS, 1% (vol/vol) Hepes (Gibco) and 1% (vol/vol) penicillin/ streptomycin (complete HBSS or cHBSS). Samples were centrifuged at 600 g for 5 min, the cell pellet was resuspended in 30% (vol/vol) Percoll (Cytiva) and centrifuged at 1590 g for 30 min. Pellet was washed in cHBSS and centrifuged at 470 g for 5 min. Finally, remaining erythrocytes were lysed using ACK buffer (100 pM EDTA, 160 mM NH4C1, 10 mM NaHCO3). To saturate Fc receptors and detect dead cells, samples were incubated respectively with FcR Block (Biolegend) and eFluor780 Fixable Viability dye (1/1000, Invitrogen) diluted in PBS for 15 min at 4°C. Then, cell suspensions were surface-labelled with the following antibodies : CD45 PerCP-Cy5.5 (30- Fl l, 1/300, BD Pharmingen), CDl lb PE-CF594 (MI/70, 1/3000, BD Horizon), CD3s BV421 (145-2C11, 1/300, BD Horizon), Ly6GBV510 (1A8, 1/200, Biolegend), Ly6C BV711 (HK1.4, 1/1800, Biolegend), MHC II I-A/I-E FITC (2G9, 1/300, BD Pharmingen), CD86 APC (GL1, 1/300, BD Pharmingen) in PBS supplemented with 0,5% (vol/vol) FBS and 0,4% (vol/vol) EDTA (Sigma-Aldrich) for 30 min at 4°C. Samples were fixed for 20 min at 4°C in 4% paraformaldehyde solution (Electron Microscopy Sciences) diluted in PBS, acquired on a BD Fortessa X20 cytometer and analyzed using FlowJo (TreeStar, vlO.7.1).

Bulk RNA sequencing. Microdissected hippocampi were disrupted with ultra-turrax® in ice cold QIAzol Lysis Reagent (Qiagen) and total RNA was purified with RNeasy Lipid Tissue Mini Kit (Qiagen). Libraries were generated with the Illumina® Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit and the IDT ILMN RNA UDI B Lig 96 Idx 96 Spl index kit (Illumina) following the manufacturer’s instructions. Briefly, RNA quantity was normalized, ribosomal RNA was depleted then RNA was fragmented and denatured. After cDNA synthesis, anchors were ligated and libraries were amplified. For differentially expressed gene analyses, raw sequencing reads were processed using nfcore/rnaseq pipeline (v3.2)^55,56. Briefly, quality of raw sequencing files was checked using fastqc (v.0.11.9) [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/], reads were trimmed using trimgalore (v.0.6.6) [https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/] to remove Illumina universal adapter sequences. Quality of trimmed reads was assessed using fastqc (v.0.11.9) and indicated complete removal of adapter sequences. Trimmed reads were aligned to GRCm39 mouse genome reference assembly and genome annotation from Ensembl (mm39, release 103) using STAR (v.2.6.1d)⁵⁷ and read quantification was performed using salmon (v.1.4.0)⁵⁸. Exploration and differential expression analysis of RNAseq data was performed using R [https://www.R-project.org/] (v.4.1.3) / RStudio (v.202202.0, build 443) and DESeq2 R package (vl.34.0)⁵⁹ using a custom script. Salmon transcript-abundance estimates were imported using the tximport R package (v 1.22.O)⁶⁰ and summarized at the gene level using the ‘salmon_tx2gene’ file generated by nfcore/rnaseq pipeline. Raw count matrix was filtered to remove lowly expressed genes. Genes having a minimum of 10 normalized reads in 3 of the 4 replicates in any of the Uninfected, Latency or Encephalitis samples were kept. Principal component analyses and hierarchical clustering using variance-stabilized normalized counts indicated that samples clustered together according to their group. Differential expression analyses were performed using DESq2::DESeq() function and extracted using DESq2: :results() function with a TfcThreshold’ parameter of 1 and an ‘alpha’ parameter of 0.05 (i.e. adjusted p-value < 0.05). Functional enrichment analyses were performed using clusterProfiler (v 4.2.2)⁶¹ R packages.

Cytokine and chemokine quantification. Serum IL- lb levels were assessed by ELISA according the manufacturer's instructions (Quantikine ELISA kit, R&D Systems). Microdissected hippocampi were homogenized with the FastPrep-24™ Classic Instrument in Lysing MatrixD tubes (MP Biomedicals) containing ice-cold cell lysis buffer (Invitrogen) supplemented with protease inhibitors (Thermo Fisher Scientific) and ImM of PhenylMethylSulfonylFluoride (PMSF, Sigma- Aldrich) solution prepared in 2-Propanol (Sigma- Aldrich). Tissue lysates were centrifuged at 15 800 g for 15 min at 4°C, and cleared supernatants were collected and stored at -80°C until multiplex immunoassay. Total protein concentration of the cleared lysate was assessed with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). IL-ip was quantified with V-PLEX Mouse IL-ip Kit (Meso Scale Discovery) following the manufacturer’s instructions.

Whole brain parasite load analysis. Hemibrains were homogenized in PBS using Potter tissue grinder, then 10% of the homogenate was used either to assess total parasite load (tachyzoite and bradyzoite parasite stages) by qPCR or to enumerate cysts by visual counting as described earlier⁴⁹. Cyst wall was labelled with rhodamine or fluorescein-conjugated Dolichos Biflorus Agglutinin (DBA, Vector Laboratories) and cysts were counted using an inverted fluorescence microscope at X20 magnification. For total parasite load quantification, genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions and a 529-bp repeat element in the T. gondii genome was amplified using the TOX9 (5^Z -AGGAGAGATATCAGGACTGTAG - SEQ ID NO:5) and TOX11 (5^Z - AGGAGAGATATCAGGACTGTAG - SEQ ID NO:6) primers⁶². The number of parasite genome per pg of brain DNA was estimated by comparison with a standard curve, established with a known number of Prugniaud (Pru) tachyzoites.

Blind-coding. Investigators who obtained data were blinded to infection and treatment of cell cultures.

Statistical analysis. GraphPad Prism software (v8.4.0) and R studio (v4.1.0) were used for statistical analysis. Data normal distribution was determined with Agostino & Pearson normality test, and the Bartlett-test was used to verify equality of variance. Welch’s correction was applied when the variances were not equal. Parametric or non-parametric tests and corrections for multiple comparisons were described in the legend of each figure. To assess whether differences observed in strategy types used during the Barnes maze were statistically significant between infected and uninfected groups, a Freeman-Halton extension of the Fisher’s exact test was performed on strategies (mixed and spatial groups were fused to allow appropriate use of the test since mixed strategy is partly spatially-driven). Null hypotheses were rejected at the 0.05 level. Results

Chronic Toxoplasma gondii infection impairs spatial consolidation and precision memory. First, to fully characterize the impact of T. gondii infection on cognitive behaviors, we performed a battery of behavioral tests on mice infected with either of two genetically- engineered T. gondii lines derived from the same type II Prugniaud strain: the T. gondii. SAG1- OVA line, which causes toxoplasmic encephalitis in mice of C57BL/6J background, and the T. gondii. GRA6-OVA line, which leads to latent infection characterized by lower brain parasite load (data not shown) due to effective CD8+ T cell-mediated parasite immune surveillance in the CNS¹⁷. Behavioral testing was performed once all infected mice had recovered from the acute phase of the infection and reached back 90% of their initial weight (data not shown). To test the learning and memory capacities of the mice upon T. gondii infection, mice were challenged in the Barnes maze task. In the training component of the Barnes maze, all groups were able to learn the task and improved over time, since they all ran shorter distances and committed less and less errors prior to finding the target hole over time (Figure 1A-B). Yet, mice with encephalitis were slower to improve their performances as they visited more error holes (Figure IB) and adopted less efficient, non-spatial, search strategies of the target hole (Figure 1C-D). Nonetheless, after 5 days of training, all groups had reached similar performances, allowing us to test the recall of spatial memory in a probe trial set 24h after the last training (Figure 1E-F). Mice from all groups displayed preserved approximate memory of the target hole, since they spent significantly more time in the target quadrant where the exit hole used to be, compared with the time spent in the other 3 quadrants (Figure IE). However, mice with encephalitis displayed less precision in visiting the area as indicated by fewer visits of the exit area during the probe test compared to uninfected mice (Figure IF). Interestingly, latently infected mice also visited significantly less the exit area than uninfected mice (Figure IF), suggesting discrete impairment in memory retrieval upon both encephalitis and latent T. gondii infection. In order to better dissect the regional network mechanisms involved in these impairments that are common to the latency and encephalitis contexts, we performed two object-based tests: the novel object recognition test (NOR, Figure 1G-H) and the object location test (OL, Figure 1I-J). In the experimental conditions used for these tests, the NOR assesses memory retrieval and involves cortical areas of the brain, whereas the OL task evaluates the consolidation of spatial memory and critically relies upon the function of the hippocampus. Mice from all groups spent the same amount of time exploring objects (Figure 1G-I), and properly discriminated a new object compared to an already explored one, suggesting intact cortical circuits despite T. gondii infection (Figure 1H). Yet, T.gondii- infected mice, regardless of disease severity, showed impaired consolidation of spatial memory since they were unable to discriminate the moved object (Figure 1J). Together, these data indicate that T.gondii-infected mice with either encephalitis or latency are impaired in spatial memory retrieval and consolidation, which depend on the function of the hippocampus.

Chronic Toxoplasma gondii infection upregulates IL-1 dependent pathways which are responsible for the cognitive impairment. To start investigating the cellular mechanisms which may lead to impaired consolidation of spatial memory, we determined the changes in numbers and activation state of non-neuronal cell populations in the hippocampus (e.g. astrocytes, microglia, monocytes, granulocytes and T cells) by immunohistofluorescence (data not shown) and/or flow cytometry (data not shown). Latent T. gondii infection and encephalitis conditions differed in terms of parasite loads as assessed by counting the number of cysts or quantifying the parasite genome in the brain (parasite burden is higher in encephalitis compared to latent T. gondii, data not shown). The numbers of astrocytes, microglia, monocytes, neutrophils/granulocytes, and T cells, as well as the activation of microglia (measured by MHC class II and CD86 surface expression levels), were all increased in the hippocampus of infected mice compared to uninfected controls (data not shown). Despite a clear trend for a larger increase in the encephalitic mice, the differences between the encephalitis and latency conditions did not reach statistical significance. In conclusion, both models of chronic T. gondii persistence in the brain lead to immune cell infiltration, astrogliosis, microgliosis and microglia activation. To gain insights into the cytokine-related pathways that are dysregulated during chronic T. gondii infection and may cause behavioral abnormalities, we analyzed gene expression changes in the hippocampus of uninfected vs. infected mice by bulk RNA-sequencing, and we used these data to infer pathways significantly enriched in both latency and encephalitic mice vs. uninfected mice. When filtering for significantly enriched pathways related to interleukins or interferons, we found pathways linked to production and response to IFN-gamma, type I IFN, IL-1, IL-10 and IL-27 (data not shown). Given i) that IL- l³⁰’⁴⁴ and IL-l-related cytokines (e.g. IL-33 33,45) have been suggested to modulate hippocampal function, ii) that the IL-1 receptor is constitutively expressed in hippocampal neurons of the dentate gyrus⁴⁶ and iii) that IL-1 signaling does not seem to be required for parasite control during chronic stage⁴⁷, we decided to further explore the role of brain IL-1- signaling in latent T.gondii-induced behavioral abnormalities. To this aim, we implemented a transgenic mouse model, wherein IL- 1 -dependent signaling can be suppressed in adult excitatory neurons by conditional CaMKIIa-restricted⁴⁸ and tamoxifen-inducible knockout⁴⁹ of the receptor for IL-1 (IL-1R1). Upon treatment with tamoxifen, CaMKIIa-CRE-ERT2: Illrlflox/flox mice loose the expression of IL-1R1 in excitatory neurons (Illrl^neuronKO mice). We confirmed that the IL- lb cytokine is more abundant in hippocampal protein extracts from latently infected compared to uninfected mice, and we observed that the absence of IL-1R1 in neurons did not change this level (Figure 2A). Despite the loss of IL-1 signaling in excitatory neurons, H_ri^neuronKO_mice displayed normoactive behavior, unmodified aversion to risk, and similar capacities of learning, consolidating and retrieval of memory compared to tamoxifen- treated Cre-negative control littermates (data not shown). Since latent T. gondii infection causes impaired consolidation of spatial memory in the OL test, we tested Illrl^WT and U 1 r 1^neuronKO mice chronically infected by latent T. gondii in the same OL task (Figure 2B-C). We confirmed that latent T.gondii-infected Illrl^WT mice were impaired in the OL task compared to uninfected littermates. Importantly, Illrl^neuronKO mice displayed intact spatial consolidation in the OL task. Weight loss and brain parasite burden were unaffected by this conditional knockout (Figure 2D-E). Hence, suppressing IL-1R1 signaling in excitatory neurons is sufficient to prevent the deleterious impact of chronic T. gondii infection on consolidation of spatial memory. Together, our data suggest a critical contribution of IL-1 signaling in neurons to the memory deficits caused by chronic T. gondii infection.

Chronic systemic exposure to low levels of IL-lb impairs the precision and consolidation of spatial memory due to a direct effect on glutamatergic neurons. Because IL-lb can be detected in the blood and/or in the brain of many chronic neurological diseases involving low- grade neuroinflammation, such as depression, brain injury or bipolar disorders^25,30’⁵⁰'⁵², or latent T. gondii infection (see Figure 2A), we decided to explore whether the deleterious impact of latent T. gondii infection on cognitive processes could be mimicked using chronic systemic administration of low concentrations of IL-lb. In other words, we sought to test if in addition to being required, neuronal IL-1 signaling would be sufficient to impair spatial memory. To address this question, we chronically exposed wild-type mice to 5pg/kg/day of recombinant mouse IL-lb using subcutaneously implanted osmotic minipumps. These devices allow to raise the serum IL-lb levels for 5 weeks (Figure 3A). A transient and modest weight loss was observed during the first 48 hours post-surgery, which was more pronounced in IL-lb-treated mice than saline controls (data not shown). IL-lb-treated mice were slightly slower to regain weight during the first 7 days post-surgery. However, by two weeks of infusion, mice had fully recovered and were indistinguishable in terms of weight and general behavior. During the last two weeks of infusion, mice were subjected to the same battery of behavioral challenges as the T.gondii-infected mice (Figure 3). In the elevated plus maze test (EPM), IL-lb-treated mice appeared normoactive and normo-anxious (data not shown). In the training component of the Barnes maze, mice from both groups displayed fast learning abilities and performed equally well in terms of distance and numbers of errors prior to finding the exit hole. The strategies deployed by all groups to find the exit hole also evolved from poorly efficient to spatial strategies over the 5 days of training (Figure 3B, 3C, 3D). When challenged with a probe trial 24h after the last training, mice from both groups remembered the global area of the exit hole, since they spent significantly more time in the target quadrant during the trial (Figure 3E). Interestingly, however, IL-lb-treated mice explored less precisely this area, as they performed statistically less visits of the target hole than saline-treated controls (Figure 3F). As indicated by the strategies to find the target hole, although not statistically significant, IL-lb-treated mice tended to adopt less the spatial strategy to identify the target hole during the probe trial, suggesting that a component of spatial memory was impaired. In order to determine which component of the memory was impaired in IL-lb-treated mice, we performed, as for T. gondii infection, the NOR (Figure 3G) and the OL (Figure 3H) tests. Mice from all groups were able to discriminate a new object compared to a previously explored object in the NOR (Figure 3G). However, IL-lb-treated mice showed impaired consolidation of spatial memory since they were unable to discriminate the moved object in the OL test, and explored both objects equally (Figure 3H). To examine the durability of these effects, mice were challenged in the Barnes maze paradigm, one month after the end of IL- lb infusion. Both groups were able to learn and progress in their ability to find the target holes over the 5 days of training (data not shown). Right after the probe test, we confirmed that serum IL- lb levels had returned below the detection limit and were indistinguishable from the saline-treated levels (data not shown). Nonetheless, IL-lb-treated mice continued to perform less precisely, as they made significantly less visits of the target hole compared to saline-treated controls (data not shown). These results suggest that despite serum weaning from IL-lb, the effects of chronic IL-lb administration on memory are long-lasting. To establish if the impact of IL-1 signaling on cognition is neuron- intrinsic and direct, we used the CaMKIIa-CRE-ERT2: Ulrl^flox/floxmice. We implanted IL-lb- or saline- infusing osmotic minipumps subcutaneously to Illrl^WT and II i_ri^neuronKO_mice and performed behavioral analyses. First, as in C57BL6/J mice upon chronic IL-lb infusion, we confirmed that IL-lb-infused Illrl^WT mice were impaired in precision and consolidation of spatial memory in the Barnes maze and OL tests (Figure 31 and data not shown). Remarkably, U 1 r 1^neuronKO mice infused with IL-lb performed as well as saline-treated mice, showing that knocking out II lrl^m glutamatergic neurons is sufficient to prevent the impact on memory, of chronic IL-lb administered systemically. Together, these data indicate that chronic peripheral IL-lb impairs the spatial component of memory retrieval and consolidation, through a direct signaling within excitatory glutamatergic neurons of the hippocampus.

Chronic systemic exposure to low level of IL-lb induces leukocyte infiltration in the hippocampus without microglial activation. To assess how hippocampus-dependent cognitive impairments caused by chronic levels of IL-lb relate to changes in brain-resident or brain-infiltrated immune cells, we first analyzed the distribution and activation of non-neuronal cells such as astrocytes, microglia and immune cells by immunofluorescence microscopy and flow cytometry analyses. First, we confirmed that IL-lb is present in the hippocampus following chronic systemic infusion of IL-lb (data not shown). Chronic IL-lb seemed to only mildly impact microglia numbers and did not significantly impact astrocytes nor microglia activation (data not shown). However, chronic systemic low levels of IL-lb drove CNS infiltration of granulocytes, monocytes and T cells (data not shown), albeit with a lower magnitude than following T. gondii infection (data not shown). We next assessed to which extent IL-lb affects the neurogenic niche. As an endpoint study, we counted the number of newly bom doublecortin-positive neurons in the dentate gyrus of the hippocampus using immunohistochemistry analyses. We found that neither chronic IL-lb (data not shown), nor changes in neuronal IL-1R1 signaling altered the number of doublecortin-positive cells, suggesting that the observed memory impairments of IL- lb -treated mice are unlikely to be due to changes in glial cell numbers and newborn neurons.

Chronic T.gondii infection and systemic exposure to IL-lb increase DNA Double-strand breaks marker levels in the hippocampus. Because of the long-lasting impact of IL-lb on hippocampus function, we hypothesized that chronic IL-lb could act on neurons through an epigenetic mechanism. Thus, we chose to explore the role of the DNA DSB response in this process. As previously shown by us and colleagues, neuronal DSB levels can be reliably quantified by immunostainings of the phosphorylated form of the histone variant H2A.X (gH2A.X), or of 53BP1, two specific hallmarks of DSB in neurons, which form foci at the DSB sites³⁹'^41,53'⁵⁵. First, we counted the number of neurons from the dentate gyrus (DG) and the Cornu Ammonis (CA) bearing 53BP1 -positive foci (data not shown). As shown previously^53,56, we confirmed using confocal immunofluorescence microscopy that mature neurons express diffuse 53BP1 staining in their nuclei (data not shown). As part of the DSB response, 53BP1 form foci at sites of DSB, that can be identified as brighter immunofluorescent dots in the nuclei of neurons (data not shown). We found that in the context of both latent T. gondii infection (Figure 4A and data not shown) and encephalitis (Figure 4B), there was a significantly increased number of neurons bearing 53BP1 -positive foci in the DG of the hippocampus. We hypothesized that this effect may depend on chronic neuroinflammation caused by the persistent infection. Chronic IL-lb-treatment elicited a similar increase in DSB- bearing neurons in the DG and CA of the hippocampi of the mice as in the context of T. gondii infection (Figure 4C), suggesting that this cytokine may directly trigger DSB in neurons. To test this hypothesis, we established primary cultures of mouse postnatal hippocampal neurons, treated them with increasing concentrations of IL- lb for hours and analyzed the levels of gH2A.X by Western blot, since gH2A.X levels are an accurate proxy of DSB in neurons³⁹'^41,53'⁵⁵. Fifty (50) ng/ml of IL-lb were sufficient to increase by over 40% the amount of gH2A.X in the culture, compared to vehicle-treated controls (Figure 4D). At this concentration, no sign of neuronal loss was observed. Together, these data suggest that a DNA DSB response involving gH2A.X and 53BP1 is induced in hippocampal neurons of the DG and CA upon T. gondii infection and upon chronic systemic exposure to IL-lb.

H2A.X-dependent signaling critically contributes to memory deficits caused by T.gondii infection and by chronic exposure to IL-lb. We decided to test whether the induction of a H2A.X-mediated DNA DSB response plays a role in the consolidation deficits of spatial memory in mice. Because the complete knockout of genes involved in the DSB response is often associated with embryo lethality or neurodevelopmental deficits caused by genome instability⁵⁷'⁵⁹, we decided to target H2A.X expression selectively in neurons in adult animals. To this aim, we treated adult CaMKIIa-CRE-ERT2: H2ax^flox/flox mice with tamoxifen to invalidate the H2ax gene in excitatory neurons at adulthood. We verified that CaMKIIa-CRE- ERT2: H2ax^flox/flox treated with tamoxifen loose the expression of H2A.X in excitatory neurons (H2A.X^neuronKO mice, data not shown). Absence of H2A.X in excitatory neurons did not impact the general brain morphology, the number of newborn neurons or the global distribution of glial cells (data not shown). First, we implanted H2ax^neuronKO and control H2ax^WT mice with osmotic IL- lb vs saline mini-pumps as previously, and tested the mice in the OL task (Figure 5A). As expected, IL- lb impeded consolidation of spatial memory in H2ax^WT mice as indicated by the absence of preferred exploration of the moved object. Interestingly, saline treated H2ax^neuronKO mice had the same ability to consolidate memories as H2ax^WT mice exposed to saline, showing that under basal/non-inflammatory conditions, H2A.X expression in neurons is not required for the learning and the consolidation components of spatial memory. Notably, H2ax^neuronKO mice treated with chronic IL- lb were also able of memory consolidation, showing that knocking out H2A.X was protective against the deleterious impact of IL- lb on memory. Finally, we confirmed this finding in the more complex situation of T. gondii infection. In the OL task, we confirmed the T. gondii induced crippling of consolidation processes in H2ax^WT mice (Figure 5B-C) As above with IL- lb administration, we found that knocking out H2A.X in excitatory neurons was sufficient to prevent the deleterious impact of T. gondii encephalitis on consolidation of spatial memory, despite similar parasite burden (Figure 5B-C). Taken together, our results show that blockade of the H2A.X-mediated DNA DSB response is protective against the IL- 1 -induced impairment of spatial memory consolidation not only in a simplified model of IL-lb systemic infusion but also in the relevant and prevalent context of CNS infection.

CONCLUSION

Our study shows that IL-1 signaling in excitatory neurons contributes to memory impairment in two chronic pathological contexts leading both to an increase in IL-1 in the hippocampus (i.e. a brain persisting infection by T. gondii parasites, and a chronic exposure to systemic IL-lb). Additionally, we uncover that DNA DSB signaling mediated by H2A.X in neurons, is a critical mechanism involved in the IL-l-induced spatial memory deficits. We indeed found that the IL-l-induced memory impairment could be completely prevented by the invalidation of H2A.X, a histone variant involved in the DNA DSB response. This provides an unprecedented proof of concept that the IL-1 and H2A.X signaling pathways may be relevant therapeutic targets to mitigate memory dysfunction in neuroinflammatory pathologies. Table 1. List of transgenic Prugniaud and ME49 derivative strains used in this study

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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file:///H:/DOSSIERS/S/SUBERBIELLE23193/TEXTE DE BREVET/PRIORITE/DEPOT/SequencesPROJ_23193 EP.xml

Claims

CLAIMS:

1. A method of treating a subj ect suffering from cognitive deficit comprising administering to said subject a therapeutically effective amount of an inhibitor of IL- 1 -induced H2A.X signaling.

2. The method of claim 1 wherein the inhibitor of IL- 1 -induced H2A.X signaling is selected from the list consisting in an IL-1 inhibitor, an IL-1R inhibitor or an H2AX inhibitor.

3. The method of claim 1 or 2, wherein the inhibitor of IL- 1 -induced H2A.X signaling is an IL- ip inhibitor.

4. The method of claim 1 to 3, wherein the subject suffers from memory trouble.

5. The method of claim 1 to 3, wherein the subject suffers from a neuroinflammation.

6. The method of claim 1 to 5, wherein the inhibitor of IL- 1 -induced H2A.X signaling is an inhibitor of IL-1, of IL-1R or of H2AX gene expression selected from the list consisting of antisense oligonucleotide, nuclease, siRNA, shRNA or ribozyme.

7. The method of claim 1 to 5, wherein the inhibitor of IL- 1 -induced H2A.X signaling is anakinra, rilonacept canakinumab or gevokizumab.