A CSF1r inhibitor and a bone marrow cell population for the replacement of microglia and central nervous system-associated macrophages in central nervous system diseases

Abstract

The invention relates to the field of neurology, neurobiology, and neurological disorders. The invention relates to a pharmaceutical combination comprising a colony-stimulating factor 1 receptor (CSFR1R) inhibitor and a bone marrow cell population for use in the treatment of central nervous system (CNS) diseases. The invention further relates to an ex vivo method for depleting dysfunctional and / or mutant microglia and / or CNS-associated macrophages (CAM), the method comprising providing a population of cells comprising myeloid cells and treating said cells with an CSF1R inhibitor of central nervous system (CNS)-associated myeloid cells, thereby depleting dysfunctional and / or mutant microglia. The invention relates to a cell population according to the ex vivo method. The invention further relates to a pharmaceutical composition comprising the CSF1R inhibitor according to the present invention for use in the treatment or prevention of a medical condition induced by dysfunction of immune cells in a central nervous system (CNS), additionally comprising a pharmaceutically acceptable carrier.

Description

[0001] REPLACEMENT OF MICROGLIA AND CENTRAL NERVOUS SYSTEM-ASSOCIATED MACROPHAGES IN CENTRAL NERVOUS SYSTEM DISEASES

[0002] DESCRIPTION

[0003] The invention relates to the field of neurology, neurobiology, and neurological disorders.

[0004] The invention relates to a pharmaceutical combination comprising a colony-stimulating factor 1 receptor (CSFR1 R) inhibitor and a bone marrow cell population for use in the treatment of central nervous system (CNS) diseases.

[0005] The invention further relates to an ex vivo method for depleting dysfunctional and / or mutant microglia and / or CNS-associated macrophages (CAM), the method comprising providing a population of cells comprising myeloid cells and treating said cells with an CSF1 R inhibitor of central nervous system (CNS)-associated myeloid cells, thereby depleting dysfunctional and / or mutant microglia.

[0006] The invention relates to a cell population according to the ex vivo method.

[0007] The invention further relates to a pharmaceutical composition comprising the CSF1 R inhibitor according to the present invention for use in the treatment or prevention of a medical condition induced by dysfunction of immune cells in a central nervous system (CNS), additionally comprising a pharmaceutically acceptable carrier.

[0008] BACKGROUND OF THE INVENTION

[0009] Central nervous system (CNS) diseases are neurological disorders affecting the brain or spinal cord. These disorders can result from various causes, including infections, injuries, blood clots, age- related degeneration, cancer, autoimmune dysfunction, and congenital disabilities. Symptoms and treatments for CNS diseases vary widely, depending on the specific condition. Some of the most common CNS disorders include multiple sclerosis, Alzheimer's disease, epilepsy, and various psychiatric conditions. Notably, CNS tumors are the most common pediatric cancers, with brain tumors having the highest mortality rates among them. Treatments for CNS diseases range from surgery and neural rehabilitation to prescribed medications. According to a major study published in The Lancet Neurology, over 3 billion people worldwide were living with a neurological condition in 2021 , with neurological conditions now being the leading cause of ill health and disability globally.

[0010] The central nervous system (CNS) hosts organ-specific immune cells, known as microglia in the parenchyma and perivascular, meningeal, and perivascular macrophages at CNS barriers, collectively referred to as CNS-associated macrophages (CAMs). Microglia and CAMs are plastic, long-lived, and host numerous risk genes for multiple pathologies, making them the most suitable targets for modulating CNS diseases (M Prinz et al., 2021). Microglia have been implicated in causing diseases known as "microglioopathies," which can arise from mutations in genes such as CSF1 R, USP18, TYROBP, TREM2, NRROS, and HEXB. Beyond these rare monogenic disorders, genome-wide association studies (GWAS) have suggested that microglia and CAMs play a crucial role in various neuroinflammatory and neurodegenerative diseases. Dozens of loci affecting microglial phagocytosis, activation, or immunoregulation have been linked to Parkinson's disease (e.g., TREM2), Alzheimer's disease (e.g., CD33), frontotemporal dementia (e.g., GRN), schizophrenia (e.g., C4), multiple sclerosis (e.g., TNFRSF1A, IRF8), and neuropathic pain (e.g., P2X4R). Additionally, microglial dysfunction is reported in neurodevelopmental and neuropsychiatric disorders, including Rett syndrome, autism spectrum disorder, depression, and anxiety disorders.

[0011] Colony-stimulating factor 1 receptor (CSF1 R) is a tyrosine kinase transmembrane receptor encoded by the CSF1 R gene. It is the receptor for colony-stimulating factor 1 (CSF-1) and interleukin-34 (IL- 34), regulating the production, differentiation, and function of macrophages. CSF1 R signaling is critical in various diseases, including myeloid malignancies, neurodegenerative diseases, and inflammatory bone diseases. In neurobiology, CSF1 R is essential for developing and maintaining microglia and has been implicated in neurodegenerative diseases. Despite its known role, no therapeutic approaches targeting CSF1 R are available for treating neurodegenerative diseases. CSF1 R inhibitors have been explored for their potential therapeutic applications, particularly in cancer therapy, where they target tumor-associated macrophages (TAMs). However, while these inhibitors are being explored for conditions beyond cancer, none are currently known to be used for neurological diseases.

[0012] Lysosomal storage disorders (LSDs) are a subset of CNS diseases. They comprise over 50 rare genetic diseases characterized by the accumulation of toxic substances within the body's cells due to deficiencies in specific lysosomal enzymes, activator proteins, or membrane transport proteins. These deficiencies result from gene mutations responsible for encoding proteins that facilitate lysosomal function. The buildup of undegraded materials can lead to cellular and organ damage, significantly affecting various organ systems, particularly the nervous system. Examples of LSDs include Gaucher disease, Sandhoff disease, and Tay-Sachs disease. Sango et al. developed a Hexb knockout mouse model to study Sandhoff disease, which has been extensively used to describe the disease's underlying pathology. Various experimental therapeutic approaches have been tested on these mice, primarily focusing on delivering functional genes to neurons via viral vectors. However, this method fails to provide a functional gene on a CNS-wide scale. While treated mice experience delayed symptoms and prolonged survival, the disease remains fatal. In humans, no causal treatment regimen for neurodegenerative diseases, such as lysosomal storage diseases, has been established, highlighting the urgent need to develop new therapeutic tools to address the root cause of these diseases. Additionally, there are currently no effective microglia / CAM-specific therapeutic options available.

[0013] US 2023 / 398153 A1 discloses a method for replacing microglia with bone marrow-derived cells by hematopoietic stem cell transplantation (HSCT) and administration of a glial cell conditioning agent, such as a CSF1 R inhibitor, after HSCT.

[0014] Colella Pasqualina et al. (NATURE COMMUNICATIONS, vol. 15, no. 1 , 5 July 2024, XP093244837) show that bone marrow transplantation in progranulin-deficient mice conditioned with busulfan and the CSF1 R inhibitor PLX3397 restores progranulin in the brain and eyes and normalizes lipofuscin storage, proteostasis, and lipid metabolism in the brain.

[0015] Colella Pasqualina et al. (Molecular Therapy, 2023, pages 160-161 , XP093244858) reveal the use of PLX3397 and bone marrow transplants to repopulate the CNS, with the differentiation of iPSCs into tendon cells optimized by single-cell analysis.

[0016] Wu et al. (Multiple Sclerosis Journal, 30 May 2023, pages 110-110, XP093244854) describe the therapeutic effect of transplanting myeloid cells from bone marrow into the CNS after bone marrow transplantation in chronic experimental autoimmune encephalomyelitis (EAE) in a mouse model. PLX5622 is used for the temporary depletion of microglia.

[0017] Colella Pasqualina et al. (Human Gene Therapy, 14 December 2022, pages A21-A21 , XP093244859) disclose a conditioning method that enables the repopulation of macrophage and microglia niches by wild-type and genome-edited hematopoietic cells. Furthermore, the use of busulfan and PLX3397 for conditioning prior to hematopoietic cell transplantation, including the repopulation of microglia in the brain, for the treatment of neurometabolic diseases is described.

[0018] Colella Pasqualina et al. (Human Gene Therapy, 15 February 2024, pages A30-A31 , XP093244857) reveal repopulation of the brain with hematopoietic, microglia-like cells in progranulin-deficient mice treated with busulfan and PLX3397.

[0019] Thus, the prior art describes separate or temporally sequential approaches involving microglia depletion and subsequent cell transplantation, without disclosing or suggesting a pharmaceutical combination for the simultaneous treatment of central nervous system (CNS) disorders and targeted microglial niche repopulation.

[0020] Consequently, there is an unmet need for strategies to treat CNS diseases. Given the current state of the art, there remains a significant demand for advanced techniques that address the root causes of CNS diseases rather than just alleviating the symptoms.

[0021] SUMMARY OF THE INVENTION

[0022] In light of the prior art, the technical problem underlying the invention was providing alternative or improved means for strategies to treat central nervous system (CNS) diseases. The present invention seeks to provide such means while avoiding the disadvantages known in the prior art.

[0023] A further object of the invention was to provide alternative or improved means for treating CNS diseases by addressing the root causes.

[0024] Another objective of the invention was to provide alternative or improved means for treating CNS diseases with minimized side effects.

[0025] A further objective of the invention was to provide alternative or improved means for depleting and / or replacing dysfunctional and / or mutant microglia and / or CNS-associated macrophages (CAMs).

[0026] These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims. Thus, the present invention, in one aspect, relates to a pharmaceutical combination comprising a. a colony-stimulating factor 1 receptor (CSF1 R) inhibitor and b. a bone marrow cell population for use in the treatment of central nervous system (CNS) diseases.

[0027] In embodiments, the CSF1 R inhibitor is an inhibitor of immune cells.

[0028] In embodiments, the CSF1 R inhibitor is an inhibitor of central nervous system (CNS)-associated myeloid cells.

[0029] In embodiments, the CSF1 R inhibitor is an inhibitor of central nervous system (CNS)-associated macrophages (CAM) and / or microglia.

[0030] In embodiments, the CSF1 R inhibitor is an inhibitor of CNS-associated macrophages and / or precursor cells thereof, preferably monocyte-derived macrophages. In other embodiments, the CSF1 R inhibitor is an inhibitor of microglia and / or precursor cells thereof.

[0031] Advantageously, the CSF1 R inhibitor can target both, the CNS-associated macrophages and microglia.

[0032] The prior art describes separate or temporally sequential approaches involving microglia depletion and subsequent cell transplantation, without disclosing or suggesting a pharmaceutical combination comprising a CSF1 R inhibitor and a defined bone marrow-derived cell population for the simultaneous treatment of central nervous system (CNS) disorders and targeted microglial niche repopulation.

[0033] In contrast, the present invention advantageously enables a controlled and efficient replacement of dysfunctional microglia with therapeutic donor-derived cells within a single treatment regimen, thereby minimizing treatment complexity, reducing patient burden, and potentially enhancing the therapeutic outcome through optimized timing and synergy between depletion and engraftment steps.

[0034] A skilled person is capable of determining successful inhibition through an inhibitor, e.g. CSF1 R inhibitor. Methods to determine depleted microglia or CAMs may include, but are not limited to, cell counting techniques, such as microscopy and / or flow cytometry; imaging techniques, such as immunohistochemistry, immunofluorescence, and / or positron emission tomography (PET) imaging; biochemical techniques, such as Western blotting and / or ELISA; and / or genetic approaches using reporter mice.

[0035] In embodiments, the CSF1 R inhibitor depletes mutant and / or dysfunctional microglia and / or CNS- associated macrophages (CAM) in a central nervous system (CNS)-wide manner.

[0036] In embodiments, the CSF1 R inhibitor depletes mutant and / or dysfunctional microglia in a CNS-wide manner. In embodiments, the CSF1 R inhibitor depletes mutant and / or dysfunctional CNS-associated macrophages (CAMs) in a CNS-wide manner. Surprisingly, the pharmaceutical combination as disclosed enables the depletion of mutant and / or dysfunctional microglia and / or CAMs in an CNS-wide manner and the replacement of said microglia and / or CAMs with healthy microglia and / or CAMs.

[0037] To date, there are no known cures for CNS diseases, such as lysosomal storage diseases (LSDs). Even though several treatment options are available, they can be arduous and challenging for patients. These treatment options include enzyme replacement therapy (ERT), stem cell transplantation, substrate reduction therapy (SRT), and gene therapy. Other treatments manage symptoms of the disease.

[0038] Studies in Hexb knockout mice have focused on delivering functional genes to neurons using viral vectors. However, this approach does not achieve gene delivery across the entire central nervous system (CNS). As a result, while treated mice may experience prolonged survival and delayed symptoms, the disease ultimately remains fatal. In humans, no causal treatment regimen has been established, and effective microglia / CAM-specific therapies are not available.

[0039] Surprisingly, the present invention overcomes these challenges by targeting CAMS and / or microglia.

[0040] As demonstrated in the examples below, HexB-mutant microglia could be replaced with healthy microglia in a CNS-wide manner. Surprisingly, the treated mice displayed no impaired survival, with all analyzed mice surviving their genetic disorder. Additionally, treated mice were prevented from developing the severe motor symptoms observed in untreated controls.

[0041] Thus, to the knowledge of the inventors, the present invention relates to the first described pharmaceutical combination and the first functional evidence thereof to achieve the desired therapeutic effect. Alone the method to replace mutant microglia with healthy microglia in a CNS- wide manner as described herein, represents a significant and beneficial breakthrough in treating the many diseases associated with the CNS. Consequently, this invention introduces a novel technique to restore the central nervous system's endogenous immune system, involving the partial replacement of microglia / CAM through pharmacological depletion of these cells, followed by the introduction of (genetically modified) bone marrow myeloid cells.

[0042] Furthermore, the pharmaceutical combination of the present invention also enables the inhibition of genes associated with a CNS-based immune response.

[0043] In embodiments, the CSF1 R inhibitor inhibits genes associated with a central nervous system (CNS)-based immune response.

[0044] In embodiments, the genes associated with a CNS-based immune response are colony-stimulating factor 1 receptor (CSF1 R), ubiquitin-specific peptidase 18 (USP18), transmembrane immune signaling adaptor (TYROBP), triggering receptor expressed on myeloid cells 2 (TREM2), negative regulator of reactive oxygen species (NRROS), hexosaminidase A (HEXA), hexosaminidase B (HEXB), CD33, granulin (GRN), complement component 4 (C4), TNF receptor superfamily member 1A (TNFRSF1 A), interferon regulatory factor 8 (IRF8), and / or purinergic receptor P2X 4 (P2X4R), more preferably CSF1 R, USP18, HEXB, or lRF8.

[0045] In embodiments, the CSF1 R inhibitor inhibits genes associated with a microglia and / or CNS-based immune response based on mutations, wherein said genes are CSF1 R, USP18, TYROBP, TREM2, NRROS, HEXA, HEXB, CD33, GRN, C4, TNFRSF1A, IRF8, and / or P2X4R, more preferably CSF1 R, USP18, HEXB, and / or IRF8, preferably CSF1 R, USP18, HEXB, and / or IRF8.

[0046] In embodiments, the CSF1 R inhibitor inhibits genes associated with a microglia and / or CNS-based immune response, wherein said genes carry one or more mutations.

[0047] In embodiments, the CSF1 R inhibitor inhibits mutated genes associated with a microglia and / or CNS-based immune response, wherein said mutated genes are CSF1 R, USP18, TYROBP, TREM2, NRROS, HEXA, HEXB, CD33, GRN, C4, TNFRSF1A, IRF8, and / or P2X4R, more preferably CSF1 R, USP18, HEXB, and / or IRF8, preferably CSF1 R, USP18, HEXB, and / or IRF8.

[0048] Advantageously, by specifically targeting genes associated with a CNS-based immune response, as described above, the treatment of CNS diseases becomes more precise compared to targeting the entire immune system. This targeted approach can lead to more effective treatments with fewer side effects when using the pharmaceutical combination described herein.

[0049] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in one or more genes associated with the CNS-based immune response, preferably in CSF1 R, USP18, TYROBP, TREM2, NRROS, HEXA, HEXB, CD33, GRN, C4, TNFRSF1A, IRF8, and / or P2X4R, more preferably CSF1 R, USP18, HEXB, and / or IRF8.

[0050] Several genes play critical roles in CNS diseases. Surprisingly, the disclosed pharmaceutical combination has demonstrated efficacy in treating cells with mutations in these genes. This innovative approach enables targeted intervention towards these genetic factors, thereby offering potential therapeutic benefits across a spectrum of CNS disorders.

[0051] In embodiments, the CSF1 R inhibitor is selected from BLZ945, PLX5622, PLX3397, ABT-869, SC- 203877, OSI-930, JNJ-40346527, CSF1 R-IN-1 , ABSK021 , ARRY-382, and / or AZD7507, preferably BLZ945, PLX5622, and / or PLX3397.

[0052] In preferred embodiments, the CSF1 R inhibitor is a small molecule. In other embodiments, the CSF1 R inhibitor is an antibody. In embodiments, the CSF1 R inhibitor is a macromolecule, preferably an aptamer or a peptide.

[0053] It is particularly advantageous that various CSF1 R inhibitors can be used.

[0054] In embodiments, the bone marrow cell population is autologous and / or allogeneic.

[0055] Advantageously, the pharmaceutical combination described in the present invention offers the flexibility of using autologous or allogeneic bone marrow cell populations. This flexibility enhances treatment options and may improve patient outcomes. Consequently, the pharmaceutical combination as disclosed supports a personalized approach to treating diseases, preferably CNS diseases.

[0056] In embodiments, dysfunctional and / or mutant CNS-associated macrophages (CAMs) and / or microglia are treated with an CSF1 R inhibitor, thereby depleting 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% to 100% dysfunctional and / or mutant CNS-associated macrophages (CAMs) and / or microglia, preferably 70% to 100%, more preferably 80% to 100%, most preferably 100%. In embodiments, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% dysfunctional and / or mutant CNS-associated macrophages (CAMs) and / or microglia are replaced with healthy CNS-associated macrophages (CAMs) and / or microglia. The percentage of replaced CAMs or microglia may also fall within a percentage range formed between any two endpoints mentioned in the list.

[0057] In embodiments, dysfunctional and / or mutant CNS-associated macrophages (CAMs) and / or microglia are replaced with autologous and / or allogeneic bone marrow cell populations.

[0058] In embodiments, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% dysfunctional and / or mutant CNS-associated macrophages (CAMs) and / or microglia are replaced with autologous and / or allogeneic bone marrow cell populations. The percentage of replaced CAMs or microglia may also fall within a percentage range formed between any two endpoints mentioned in the list.

[0059] In embodiments, dysfunctional and / or mutant CNS-associated macrophages (CAMs) and / or microglia are replaced with genetically modified, autologous and / or allogeneic bone marrow cell populations.

[0060] In embodiments, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% dysfunctional and / or mutant CNS-associated macrophages (CAMs) and / or microglia are replaced with genetically modified, autologous and / or allogeneic bone marrow cell populations. The percentage of replaced CAMs or microglia may also fall within a percentage range formed between any two endpoints mentioned in the list.

[0061] In embodiments, the central nervous system (CNS) disease is a neurological condition and / or a mental disorder.

[0062] In embodiments, the present invention also relates to methods of treating subjects of a disease or condition mediated by CSF1 R, comprising administering the pharmaceutical composition of this disclosure to said subject.

[0063] In embodiments, the central nervous system (CNS) disease is selected from lysosomal storage disease (LSD), microgliopathies, Parkinson's disease, Alzheimer's disease, frontotemporal dementia, multiple sclerosis, Rett syndrome, autism spectrum disorder, depression, anxiety disorders, neuropathic pain, and / or schizophrenia.

[0064] In embodiments, the CNS disease is a microgliopathy. In embodiments, the CNS disease is a microgliopathy, preferably a type I microgliopathy or a type II microgliopathy.

[0065] In embodiments, a microgliopathy is caused by mutations in genes, preferably TREM2, TYROBP, and / or CSF1 R.

[0066] In some embodiments, a microgliopathy causes additional neurodegenerative diseases, preferably neurological and / or psychological disorders.

[0067] In preferred embodiments, the neurological condition is a lysosomal storage disease (LSD), preferably Sandhoff disease or Tay-Sachs disease.

[0068] In embodiments, the CNS disease is a lysosomal storage disease (LSD), preferably Sandhoff disease or Tay-Sachs disease. In embodiments, the CNS disease is a Sandhoff disease. In one embodiment, the Sandhoff disease is an infantile, a juvenile, or an adult Sandhoff disease. In embodiments, the Sandhoff disease is caused by mutations in HEXB encoding the p-subunit of p-hexosaminidase A.

[0069] In embodiments, the CNS disease is a Tay-Sachs disease.

[0070] In one embodiment, the central nervous system (CNS)-associated myeloid cells carry a mutation in one or more subunits of beta-hexosaminidase enzyme complex, preferably the alpha and / or beta subunit.

[0071] In another embodiment, a HEXA gene carries a mutation in one or more subunits of betahexosaminidase enzyme complex, preferably the alpha subunit.

[0072] In another embodiment, a HEXB gene carries a mutation in one or more subunits of betahexosaminidase enzyme complex, preferably the beta subunit.

[0073] In embodiments, a mutation of a HEXA and / or HEXB gene leads to impaired GM2 gangliosides.

[0074] In embodiments, the neurological condition is a genetic disorder, preferably GM2 gangliosidosis.

[0075] In one embodiment, the patient exhibits a deficiency in the protein GM2 activator. In another embodiment, the patient exhibits a deficiency in the enzyme beta-hexosaminidase A (Hex A). In further embodiments, the patient exhibits a deficiency in the beta-hexosaminidase B (Hex B).

[0076] In one embodiment, the patient exhibits an accumulation of the ganglioside GM2 in the nervous system.

[0077] Several techniques can be employed to determine a deficiency or accumulation of enzymes, such as HEXA, HEXB, or GM2 activator. Well-known techniques in the art comprise but are not limited to enzyme activity assays, Western Blots, immunohistochemistry, mass spectrometry and / or genetic testing.

[0078] In embodiments, the catalytic activity of the enzymes can be determined by enzymatic analysis. A skilled person is able to determine the catalytic activity of enzymes without undue effort.

[0079] In embodiments, a patient is experiencing or suffering from one of more symptoms due to a CNS disease.

[0080] In embodiments, a patient is experiencing or suffering from one or more of symptoms, preferably cognitive and / or motoric symptoms, progressive nervous system deterioration, problems initiating and controlling muscles and / or movement, early blindness, seizures, and / or spasticity.

[0081] Various symptoms are known in the art and a skilled person is able of diagnosing said symptoms with undue effort.

[0082] In another aspect, the invention relates to an ex vivo method for depleting dysfunctional and / or mutant microglia and / or CNS-associated macrophages (CAM), the method comprising a. providing a population of cells comprising myeloid cells, b. treating said cells with an CSF1 R inhibitor of central nervous system (CNS)-associated myeloid cells, thereby depleting dysfunctional and / or mutant microglia. In another aspect, the invention relates to an ex vivo method for depleting dysfunction and / or mutant microglia and / or CNS-associated macrophages (CAM), the method comprising a. providing a population of cells comprising myeloid cells, b. treating said cells with an CSF1 R inhibitor of central nervous system (CNS)-associated myeloid cells, thereby depleting dysfunctional and / or mutant microglia, wherein the myeloid cells are central nervous system (CNS)-associated macrophages (CAM) and / or microglia, wherein said cells carry a mutation in one or more genes associated with the CNS-based immune response.

[0083] In another aspect, the invention relates to an ex vivo method for depleting dysfunction and / or mutant microglia and / or CNS-associated macrophages (CAM), the method comprising a. providing a population of cells comprising myeloid cells, b. treating said cells with an CSF1 R inhibitor of central nervous system (CNS)-associated myeloid cells, thereby depleting dysfunctional and / or mutant microglia, wherein 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% to 100% of said cells are depleted, preferably 70% to 100%, more preferably 80% to 100%, most preferably 100%.

[0084] In another aspect, the invention relates to a cell population obtained according to the method of the invention.

[0085] In embodiments, the cell population obtained according to the method of invention is a healthy cell population. In embodiments, the cell populations obtained according to the method of invention are healthy CNS-associated macrophages (CAM) and / or microglia.

[0086] In embodiments, the cell population according to the invention is intended for use in the treatment of central nervous system (CNS) diseases.

[0087] In embodiments, the present invention relates to a cell population for use in the treatment of CNS diseases. In specific embodiments, the present invention relates to a cell population for use in the treatment of CNS diseases, preferably lysosomal storage disease (LSD), microgliopathies, Parkinson's disease, Alzheimer's disease, frontotemporal dementia, multiple sclerosis, Rett syndrome, autism spectrum disorder, depression, anxiety disorders, neuropathic pain, and / or schizophrenia.

[0088] In another aspect, the invention relates to a pharmaceutical composition comprising the CSF1 R inhibitor according to invention for use in the treatment or prevention of a medical condition induced by dysfunction of immune cells in a central nervous system (CNS), additionally comprising a pharmaceutically acceptable carrier.

[0089] In embodiments, the composition is administered intracerebrally, intrathecally, intravenously, or subcutaneously.

[0090] The composition described offers versatility in administration via different routes. Intracerebral and intrathecal routes are particularly suited for directly targeting the CNS, while intravenous delivery ensures systemic circulation. Subcutaneous administration provides a balanced approach between ease of use and systemic access. Intrathecal administration allows precise dosing into the cerebrospinal fluid (CSF) surrounding the CNS, facilitating targeted drug concentrations with minimized systemic exposure and potential adverse effects. These diverse delivery options enhance patient comfort and compliance, enabling tailored treatment strategies based on individual requirements.

[0091] The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the structural and / or functional features, including functional properties and beneficial technical effects, of the pharmaceutical combination described herein. The features disclosed in the context of the pharmaceutical combination also apply to and are considered disclosed in the context of the ex vivo method, the cell population, the pharmaceutical composition, and vice versa. Any features disclosed in any further aspects of the invention, such as the ex vivo method, the cell population, the pharmaceutical composition, are also considered disclosed in the context of the pharmaceutical combination and vice versa.

[0092] DETAILED DESCRIPTION OF THE INVENTION

[0093] The present invention relates to a pharmaceutical combination comprising a colony-stimulating factor 1 receptor (CSF1 R) inhibitor and a bone marrow cell population for use in the treatment of central nervous system (CNS) diseases.

[0094] All words and terms used herein shall have the same meaning commonly given to them by the person skilled in the art unless the context indicates a different meaning. All terms used in the singular shall include the plural of that term and vice versa.

[0095] Pharmaceutical combination

[0096] According to the present invention, a “pharmaceutical combination” is the combined provision of a colony-stimulating factor 1 receptor (CSF1 R) inhibitor and a bone marrow cell population.

[0097] In one embodiment, the pharmaceutical combination as described herein is characterized in that the CSF1 R inhibitor is in a pharmaceutical composition in admixture with a pharmaceutically acceptable carrier. In embodiments, the bone marrow cell population is in a separate pharmaceutical composition in admixture with a pharmaceutically acceptable carrier. The pharmaceutical combination of the present invention can, therefore, in some embodiments, relate to the provision of two separate compositions or dosage forms in proximity to each other. The combined agents are not required to be present in a single composition.

[0098] In embodiments, the CSF1 R inhibitor and the bone marrow cell population is administered sequentially. The administration duration and doses of the pharmaceutical combination may be dependent on the severity of the disease.

[0099] In embodiments, the pharmaceutical combination of the present invention restores CNS homeostasis.

[0100] Colony-stimulating factor 1 receptor (CSF1 R), also known as "macrophage colony-stimulating factor receptor (M-CSFR)" or "CD115", is a cell-surface protein encoded by the CSF1 R gene. It acts as a receptor for colony-stimulating factor 1 (CSF-1) and interleukin-34 (IL-34). Predominantly expressed in myeloid cells, CSF1 R signaling plays crucial roles in the survival, proliferation, and differentiation of various myeloid cell types. Its activation is regulated by alternative promoters and transcription factors, impacting tissue-specific expression. Structurally, CSF1 R is a tyrosine kinase transmembrane receptor belonging to the CSF1 / PDGF receptor family. Upon ligand binding, it undergoes dimerization and autophosphorylation, initiating downstream signaling cascades essential for myeloid cell function. CSF1 R signaling is implicated in numerous diseases, including cancer, neurodegeneration, and inflammatory bone disorders. Additionally, it influences non-myeloid cells, such as neural progenitor cells, contributing to neurogenesis and neurological disorders. In microglia, CSF1 R signaling plays a pivotal role in development, survival, and maintenance, influencing neuroinflammatory responses. During embryonic development, CSF1 R signaling facilitates the migration of microglia precursor cells to the brain. Subsequently, in perinatal stages, microglia aid in synaptic pruning, which is crucial for refining neuronal circuits. In adulthood, CSF1 R is essential for microglial proliferation and survival. Dysregulation of CSF1 R signaling in microglia is implicated in neurodegenerative diseases, affecting neuronal function and viability.

[0101] According to the present invention, an “inhibitor” in the context of a “CSF1 R inhibitor” is considered to be any agent, substance, compound, molecule, or other means that results in slowing, repressing, blocking, or otherwise interfering with or negatively affecting the activity, function, expression, or signaling that said target induces, performs, or exhibits in the absence of the inhibitor. The terms “agent,” “substance,” “compound,” and “molecule” can be used interchangeably.

[0102] For example, the inhibitor of the present invention may comprise an affinity reagent, antibody, or antigen-binding fragment thereof binding CSF1 R or comprise an antisense or interfering nucleic acid molecule, such as short interfering RNA (siRNA), targeting CSF1 R, or targeted protein degradation (TPD), such as proteolysis-targeted chimera (PROTAC) molecules. Preferred inhibitors are those described herein.

[0103] An inhibitor of CSF1 R may affect CSF1 R function, CSF1 R expression (transcription or translation) and / or CSF1 R -mediated signaling, either directly or indirectly.

[0104] CSF1 R inhibitors relate to an established class of inhibitors as used, for example, to control differentiation and maintenance of most tissue-resident macrophages and bone-resorbing osteoclasts (J Wen et al, 2022) and in cancer (T Fujiwara et al., 2021).

[0105] The means for determining a CSF1 R inhibitor are established in the art.

[0106] Examples of CSF1 R inhibitors include but are not limited to BLZ945, PLX5622, PLX3397, ABT-869, SC-203877, OSI-930, JNJ-40346527, CSF1 R-IN-1 , ABSK021 , ARRY-382, and / or AZD7507, preferably BLZ945, PLX5622, and PLX3397.

[0107] In embodiments, the CSF1 R inhibitor impairs microglia and / or CAM development and / or maintenance.

[0108] In embodiments, the CSF1 R inhibitor impairs microglia fate specification.

[0109] In embodiments, the CSF1 R inhibitor impairs microglial signature genes, such as P2Y purinoceptor 12 (P2ry12) and / or transmembrane protein 119 (Tmem119).

[0110] CNS and CNS-associated cells The central nervous system (CNS) comprises the brain and spinal cord, each with distinct structures and functions. The brain consists of two hemispheres responsible for higher cognitive functions, emotion, and sensory processing. Deeper brain structures like the basal ganglia and thalamus regulate motor coordination and sensory relay, respectively. The hypothalamus governs homeostatic functions such as appetite and temperature regulation. The brainstem, including the medulla, pons, and midbrain, controls vital functions like breathing, heart rate, and arousal. The cerebellum coordinates voluntary movements and balance. Additionally, the spinal cord relays sensory information to the brain and transmits motor commands from the brain to the body. Thus, these CNS components integrate sensory input, coordinate motor responses, regulate physiological functions, and mediate complex cognitive processes essential for adaptation and survival.

[0111] Myeloid cells, encompassing granulocytes and monocytes, originate from hematopoietic stem cells in the bone marrow. These cells are pivotal components of the innate immune system, distinguished from lymphoid cells by their role in immediate defense rather than adaptive immunity. Granulocytes, such as neutrophils, are swiftly recruited to infection sites, where they execute phagocytosis and release inflammatory cytokines. Monocytes, upon differentiation into macrophages or dendritic cells, contribute to antigen presentation and tissue homeostasis across various organs like the liver (Kupffer cells), lungs (alveolar macrophages), and skin (Langerhans cells). Myeloid cells exhibit high turnover rates and are crucial for combating pathogens and maintaining tissue integrity, with abnormalities in their function potentially leading to hematologic disorders like leukemia.

[0112] CNS-associated macrophages (CAMs), also known as border-associated macrophages (BAMs), comprise a specialized group of innate immune cells located at interfaces within the central nervous system (CNS). These include perivascular macrophages (PVMs), subdural leptomeningeal macrophages (MMs), and choroid plexus macrophages. Unlike microglia, which are found exclusively in the CNS parenchyma, CAMs are positioned at CNS borders, where they regulate the delicate balance between CNS isolation and controlled interaction with the periphery. Emerging evidence has revealed that CAMs, along with microglia, originate exclusively from embryonic yolk sac progenitors, which colonize the CNS during early development. While microglial precursors seed the CNS parenchyma, CAM precursors migrate to CNS interfaces such as the leptomeninges and choroid plexus, giving rise to subtypes like leptomeningeal macrophages (MnM<t>) and choroid plexus macrophages (cpM<t>). These CAM populations exhibit distinct transcriptional profiles and functional roles in health and disease. CAMs are implicated in regulating cerebrospinal fluid dynamics, responding to neuroinflammation, and contributing to neurodegenerative and cerebrovascular diseases. For instance, in Alzheimer's disease (AD) and Parkinson's disease (PD), CAMs are involved in the clearance of protein aggregates like Ap plaques and a-synuclein, and their depletion studies have shown significant impacts on disease pathology in mouse models.

[0113] As used herein, “dysfunctional CAMs” include CAMs that exhibit an abnormal phenotype and / or function due to infection, damage, and / or degeneration.

[0114] Furthermore, as used herein, the term “CAMs” may also include PVMs, MMs, and choroid plexus macrophages.

[0115] In one embodiment, the CSF1 R inhibitor targets CNS-associated macrophages located in CNS interfaces, preferably in the meninges, perivascular space, and / or choroid plexus. In embodiments, the CSF1 R inhibitor targets CNS-associated macrophages. In specific embodiments, the CSF1 R inhibitor targets perivascular macrophages (PVMs), subdural leptomeningeal macrophages (MMs), and / or choroid plexus macrophages.

[0116] Microglia are resident immune cells of the central nervous system (CNS) responsible for maintaining tissue homeostasis, surveillance, and immune defense. They originate from the yolk sac and are distributed throughout the brain and spinal cord. Microglia exist in various forms, including ramified, reactive, non-phagocytic, phagocytic, amoeboid, gitter cells, perivascular, and juxtavascular. These cells play multiple roles, including scavenging cellular debris, phagocytosing foreign materials, regulating extracellular signaling, presenting antigens, exerting cytotoxic effects, and promoting tissue repair. Plaque-associated microglia (PAMs) are a distinct population of myeloid cells attracted to insoluble Amyloid p (Ap) plaques in the brains of Alzheimer's disease (AD) patients. They form dense clusters around these plaques and undergo functional and morphological transformations. PAMs express known myeloid markers shared among macrophage progeny and microglia, but their molecular signature has only been indirectly inferred. These cells are characterized by specific disease-associated gene expression signatures and play critical roles in plaque formation, compaction, and neuroinflammation. In contrast, non-plaque-associated microglia (nonPAMs) exhibit only minor morphological alterations compared to control microglia. However, both PAMs and nonPAMs display significant temporal diversity in microglial morphology during AD progression.

[0117] As used herein, “dysfunctional microglia” include microglia that exhibit an abnormal phenotype and / or function due to infection, damage, and / or degeneration.

[0118] Furthermore, as used herein, the term “microglia” may also include “microglia precursors”.

[0119] In embodiments, dysfunctional microglia trigger CNS diseases, more preferably neurodegenerative diseases.

[0120] In certain embodiments, microglia are disease-associated microglia. In embodiments, the CSF1 R inhibitor targets disease-associated microglia. In further embodiments, the CSF1 R inhibitor depletes disease-associated microglia in a CNS-wide manner.

[0121] In certain embodiments, microglia are plaque-associated microglia. In embodiments, the CSF1 R inhibitor targets plaque-associated microglia. In further embodiments, the CSF1 R inhibitor depletes plaque-associated microglia in a CNS-wide manner.

[0122] In one embodiment, the CSF1 R inhibitor targets microglia located in the CNS parenchyma.

[0123] Genes associated with a CNS-based immune response

[0124] Ubiquitin-specific peptidase 18 (USP18), also known as UBP43, is a type I interferon receptor repressor and an isopeptidase encoded by the USP18 gene. It is induced by immune responses to type I and III interferons and serves as a negative regulator of type I interferon signaling. Loss of USP18 results in heightened sensitivity to type I interferons, leading to severe autoinflammatory disease due to disrupted signal regulation. USP18 is also a deubiquitinating enzyme that removes ISG15 conjugates from various protein substrates, a process known as delSGylation. Additionally, USP18 influences HIV replication by downregulating p21 protein expression and affects tumor progression by modulating macrophage polarization. USP18-deficiency, a rare immunodeficiency, is characterized by severe inflammation due to uncontrolled type I interferon activity and can be treated with Janus kinase inhibitors. USP18 also affects tumor-associated macrophages by modulating the CSF1 R. Deletion of USP18 in myeloid cells enhances the interaction between the ubiquitin E3 enzyme NEDD4 and CSF1 R, increasing the ubiquitination and subsequent proteasomal degradation of CSF1 R. This downregulation of CSF1 R shifts macrophages towards a pro-inflammatory phenotype, enhancing anti-tumor immune responses.

[0125] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in USP18.

[0126] In embodiments, the CSF1 R inhibitor inhibits USP18-mutated cells, preferably USP18-mutated myeloid cells.

[0127] TYRO protein tyrosine kinase-binding protein (TYROBP) is an adapter protein encoded by the TYROBP gene in humans. It contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain and participates in signal transduction by associating with membrane glycoproteins such as the killer cell immunoglobulin-like receptor (KIR) family. TYROBP interacts with zeta-chain-associated protein kinase 70 kDa (ZAP-70) and spleen tyrosine kinase (SYK), playing roles in bone modeling, brain myelination, and inflammation. Mutations in TYROBP are linked to polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL) or Nasu-Hakola disease, which presents with early-onset dementia. TYROBP and its receptor, triggering receptor expressed on myeloid cells 2 (TREM2), are crucial for osteoclast differentiation and function.

[0128] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in TYROBP.

[0129] In embodiments, the CSF1 R inhibitor inhibits TYROBP-mutated cells, preferably TYROBP-mutated myeloid cells.

[0130] Triggering receptor expressed on myeloid cells 2 (TREM2) is a membrane protein encoded by the TREM2 gene. It forms a receptor signaling complex with TYROBP and is expressed on various immune cells, including macrophages, microglia, and osteoclasts. TREM2 plays a crucial role in immune response, inflammation regulation, and phagocytosis of apoptotic cells. It is implicated in several diseases, such as polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL) and Alzheimer's disease, due to its role in microglial function and inflammatory response modulation. TREM2 interacts closely with the CSF1 R, influencing microglial activation and survival, suggesting a shared pathway in neurodegenerative diseases.

[0131] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in TREM2.

[0132] In embodiments, the CSF1 R inhibitor inhibits TREM2-mutated cells, preferably TREM2-mutated myeloid cells.

[0133] NRROS (negative regulator of reactive oxygen species) is a leucine-rich repeat-containing transmembrane protein primarily located in the endoplasmic reticulum of myeloid cells, including microglia. It regulates ROS production by interacting with and facilitating the degradation of NOX2, limiting excessive ROS generation to prevent tissue damage during inflammatory responses. NRROS is essential for the early development and maintenance of homeostatic microglial populations, playing a role in the processing and activation of TGFp. NRROS mutations are associated with chronic granulomatous disease, neurodegenerative disorders, and microgliopathies. NRROS interacts with CSF1 R, with studies indicating that disruption in CSF1 R expression affects the regulation of ROS-related genes, including NRROS.

[0134] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in NRROS.

[0135] In embodiments, the CSF1 R inhibitor inhibits NRROS-mutated cells, preferably NRROS-mutated myeloid cells.

[0136] Hexosaminidase A (HEXA) is an enzyme encoded by the HEXA gene on chromosome 15. It is part of the glycosyl hydrolase 20 family and functions with the GM2 activator protein to degrade GM2 gangliosides and other molecules with terminal N-acetyl hexosamines. HEXA is a heterodimer with an alpha subunit from the HEXA gene and a beta subunit from the HEXB gene. Mutations in HEXA lead to Tay-Sachs disease, characterized by the accumulation of GM2 gangliosides in neurons. The alpha subunit contains key residues, such as Arg-424, that enable it to hydrolyze GM2 gangliosides.

[0137] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in

[0138] HEXA.

[0139] In embodiments, the CSF1 R inhibitor inhibits HEXA-mutated cells, preferably HEXA-mutated myeloid cells.

[0140] Hexosaminidase B (HEXB) is the beta subunit of the lysosomal enzyme beta-hexosaminidase, which, together with the GM2 activator protein, degrades GM2 gangliosides and other molecules with terminal N-acetyl hexosamines. Both beta-hexosaminidase A and B are composed of alpha and beta subunits from separate genes. Mutations in HEXB lead to Sandhoff disease, a type of GM2 gangliosidosis characterized by GM2 ganglioside accumulation in neurons. HEXB mutations cause neurodegenerative disorders. Studies have shown that HEXB interacts with HEXA and gangliosides, and its expression is regulated alongside other microglia markers like P2ry12 and Tmem119. In relation to CSF1 R, HEXB's expression pattern can be influenced by microglial function and regulation, indicating its involvement in microglial activity and potentially linking HEXB expression to CSF1 R-dependent pathways.

[0141] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in

[0142] HEXB.

[0143] In embodiments, the CSF1 R inhibitor inhibits HEXB-mutated cells, preferably HEXB-mutated myeloid cells.

[0144] CD33 is a transmembrane receptor predominantly expressed on myeloid lineage cells, although it can also be found on some lymphoid cells. Belonging to the SIGLEC family of lectins, CD33 binds sialic acid residues via its extracellular immunoglobulin domains. It contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytosolic portion, which, upon phosphorylation, inhibit cellular activities like phagocytosis by recruiting SHP phosphatases. Clinically, CD33 is targeted by therapeutic agents such as gemtuzumab ozogamicin for treating acute myeloid leukemia. Moreover, CD33 serves as a marker for monocytes and has implications in diseases like Alzheimer’s disease, where it plays a role in microglial activation.

[0145] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in CD33.

[0146] In embodiments, the CSF1 R inhibitor inhibits CD33-mutated cells, preferably CD33-mutated myeloid cells.

[0147] Granulins (GRN) are a family of secreted peptides derived from the precursor protein progranulin. This 88 kDa protein undergoes cleavage in the lysosome to produce active 6 kDa granulin peptides such as granulin A, granulin B, and others. Each granulin peptide consists of 60 amino acids and is characterized by its cysteine-rich structure capable of forming multiple disulfide bonds. Granulins are critical in various physiological processes, including cell growth regulation, inflammation, wound healing, and lysosomal function. They interact with receptors on cell surfaces, influencing signaling pathways like MAPK / ERK and PI3K / Akt. Granulins are also implicated in diseases such as frontotemporal dementia and neuronal ceroid lipofuscinosis, where mutations in the GRN gene lead to their deficiency.

[0148] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in Granulin.

[0149] In embodiments, the CSF1 R inhibitor inhibits Granulin-mutated cells, preferably Granulin-mutated myeloid cells.

[0150] Complement component 4 (C4) is a protein involved in the human complement system. Originating from the human leukocyte antigen (HLA) system, C4 is crucial for linking antibody-antigen complexes to effector proteins of the innate immune response. The C4 protein undergoes proteolytic cleavage into three chains: p, a, and y. The a-chain, in particular, contains the C4d fragment involved in immune response modulation. Variations in the C4 gene, such as copy number variations and polymorphisms, are associated with disease susceptibility, including systemic lupus erythematosus, type I diabetes mellitus, and schizophrenia. C4 is part of the major histocompatibility complex (MHC) class III region on chromosome 6 and exists in multiple haplotypes, contributing to genetic diversity and influencing disease risk.

[0151] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in C4.

[0152] In embodiments, the CSF1 R inhibitor inhibits C4-mutated cells, preferably C4-mutated myeloid cells.

[0153] TNFRSF1A, also known as tumor necrosis factor receptor 1 (TNFR1), belongs to the TNF receptor superfamily and is a pivotal mediator in the immune response. This receptor exists in membranebound and soluble forms, both of which interact with TNFa, influencing diverse cellular processes such as inflammation, apoptosis, and cell survival. Activation of membrane-bound TNFR1 triggers signaling cascades involving NF-KB and apoptotic pathways through interactions with adaptor proteins like TRADD and TRAF2. Proteolytic cleavage releases a soluble form of TNFR1 , which modulates TNFa levels to mitigate inflammation. Mutations in TNFRSF1A are linked to disorders like TRAPS and potentially impact conditions such as multiple sclerosis. TNFRSFIA's association with CSF1 R, particularly in MSCLCs, underscores its role in immune responses and disease pathology.

[0154] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in TNFRSF1A.

[0155] In embodiments, the CSF1 R inhibitor inhibits TNFRSFIA-mutated cells, preferably TNFRSF1A- mutated myeloid cells.

[0156] Interferon regulatory factor s (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a transcription factor essential for the differentiation and function of myeloid lineage cells. It belongs to the IRF family and contains a conserved DNA-binding domain that enables it to bind to the IFN-stimulated response element (ISRE), regulating genes induced by type I interferons such as IFN-a and I FN-p. IRF8 plays critical roles in various cellular processes, including apoptosis regulation in myeloid cells, and has been implicated in conditions like chronic myelogenous leukemia (CML). Additionally, IRF8 governs the differentiation of dendritic cells and is integral to the development of tissue-specific macrophage populations such as microglia in the central nervous system. Its interaction with transcription factors like PU.1 and its regulatory role in immune response genes underscores its importance in cellular homeostasis and disease pathology. IRF8 is linked to CSF1 R through its expression in monocyte progenitors and its involvement in the gene regulatory networks associated with myeloid cell differentiation and function.

[0157] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in IRF8.

[0158] In embodiments, the CSF1 R inhibitor inhibits IRF8-mutated cells, preferably IRF8-mutated myeloid cells.

[0159] P2X4 receptor (P2X4R) is an ionotropic purinergic receptor belonging to the P2X receptor family, which forms trimeric complexes that function as ATP-gated cation channels. Unlike other P2X receptors, P2X4R exhibits high calcium permeability and is expressed on cell surfaces and lysosomal compartments across various tissues, including nervous systems, glands, smooth muscle, and fat cells. It plays crucial roles in cellular depolarization, synaptic strengthening, and inflammasome activation. Notably, P2X4R is implicated in neuropathic pain through microglial activation, where it mediates ATP-induced release of brain-derived neurotrophic factor (BDNF) and facilitates neuronal hyperexcitability via interaction with TrkB receptors. Moreover, P2X4R expression is regulated by CSF1 / CSF1 R signaling, which enhances its synthesis and subsequent BDNF release, highlighting its involvement in inflammatory responses and potential therapeutic strategies for neuroinflammatory diseases.

[0160] In embodiments, the central nervous system (CNS)-associated myeloid cells carry a mutation in P2X4R.

[0161] In embodiments, the CSF1 R inhibitor inhibits P2X4R-mutated cells, preferably P2X4R-mutated myeloid cells.

[0162] Central nervous system (CNS) diseases

[0163] The terms “disorder,” “disease,” or “medical condition” as used herein, can be used interchangeably. As used herein, “central nervous system (CNS) diseases” are neurological disorders that affect the structure or function of the brain or spinal cord, which together comprise the CNS. These disorders can be caused by infections, trauma, blood clots, age-related degeneration, cancer, autoimmune dysfunction, and birth defects. Symptoms vary widely and can include persistent headaches, pain, concentration issues, memory loss, muscle weakness, tremors, seizures, and paralysis. Examples of CNS diseases include Alzheimer's, Parkinson's, Huntington's, multiple sclerosis, meningitis, epilepsy, and brain tumors. Treatment options range from surgery and medication to neural rehabilitation.

[0164] As used herein, a “neurological condition” is any condition that affects the brain, spinal cord, and / or nerves and includes over 600 known disorders. They are categorized into sudden onset conditions (e.g., stroke, traumatic brain injury), intermittent conditions (e.g., epilepsy, migraine), progressive conditions (e.g., Parkinson's disease, dementia), and stable conditions with changing needs (e.g., Tourette's syndrome, narcolepsy). Neurological disorders result from structural, biochemical, or electrical abnormalities and can cause symptoms like paralysis, seizures, and pain. Causes include genetic disorders, infections, and environmental factors. Interventions range from preventive measures and therapies to medications and surgeries.

[0165] As used herein, a “mental disorder,” also known as a mental illness, mental health condition, or psychiatric disability, is a behavioral or mental pattern that causes significant distress or impairment in personal functioning. It is characterized by a clinically significant disturbance in an individual's cognition, emotional regulation, or behavior, often within a social context. These disturbances can manifest as single episodes, be persistent, or show relapsing-remitting patterns. Types of mental disorders vary widely, with common examples including but not limited to depression, bipolar disorder, schizophrenia, anxiety disorders, and neurodevelopmental disorders like ADHD and autism spectrum disorder. The exact causes are often unclear and may involve complex genetic, biological, environmental, and psychological interactions. Mental disorders are typically diagnosed by mental health professionals through various methods, including observation and psychometric tests. Treatments can include psychotherapy, medication, lifestyle changes, social interventions, and peer support, lysosomal storage disease (LSD), Parkinson's disease, Alzheimer's disease, frontotemporal dementia, multiple sclerosis, Rett syndrome, autism spectrum disorder, depression, anxiety disorders, neuropathic pain, and / or schizophrenia

[0166] Lysosomal storage diseases (LSDs) encompass a diverse group of over 70 rare inherited metabolic disorders characterized by defects in lysosomal function, which lead to the accumulation of substances inside cells. These disorders result from deficiencies in enzymes responsible for breaking down complex molecules within lysosomes. This accumulation, termed "storage," affects various organs and tissues, manifesting in symptoms that can include developmental delays, movement disorders, seizures, and organ enlargement. Examples of LSDs include but are not limited to Gaucher disease, Fabry disease, Niemann-Pick disease, Tay-Sachs disease, Sandhoff disease, and mucopolysaccharidoses like Hurler syndrome. Most LSDs are inherited in an autosomal recessive manner, although a few follow X-linked recessive patterns. Current treatments, such as enzyme replacement therapy, substrate reduction therapy, and experimental gene therapies, aim to alleviate symptoms and slow disease progression, but cures remain elusive for many LSDs. Sandhoff disease, also known as Sandhoff’s disease, is a rare lysosomal genetic disorder characterized by a deficiency in beta-hexosaminidases A and B enzymes, essential for breaking down GM2 ganglioside and other glycolipids in the nervous system and visceral tissues. This deficiency leads to the accumulation of these substances, causing progressive neurodegeneration. Clinically, Sandhoff disease manifests in three forms: classic infantile, juvenile, and adult late onset. The classic infantile form is the most severe, presenting symptoms such as developmental regression, muscle weakness, seizures, and cherry-red spots in the retina, typically leading to death by age three. Juvenile and adult forms show milder symptoms and a more variable disease course, affecting motor function and cognitive abilities. Currently, there is no cure for Sandhoff disease, and treatment focuses on managing symptoms and providing supportive care.

[0167] Tay-Sachs disease (TSD) is a genetic disorder characterized by progressive neurodegeneration due to mutations in the HEXA gene on chromosome 15, which encodes the alpha-subunit of betahexosaminidase A (Hex A), an enzyme essential for breaking down GM2 ganglioside in lysosomes. This autosomal recessive condition leads to the accumulation of GM2 ganglioside within neurons, causing cellular toxicity and, eventually, neuronal death. TSD manifests in several forms: infantile, juvenile, and late-onset / adult, each differing in age of onset and severity. Infantile TSD, the most severe form, typically presents between 3 to 6 months of age with rapid neurological decline, including loss of motor skills and vision, seizures, and eventual death by age 4. Juvenile TSD, less common and less severe, starts between 2 and 10 years and leads to death by early adolescence. Late-onset TSD, the rarest form, begins in adolescence or adulthood with variable progression and survival into adulthood. Diagnosis involves enzyme activity assays and genetic testing, while management focuses on symptom relief and supportive care.

[0168] In embodiments, the patient is experiencing or suffering from symptoms due to the CNS diseases as described herein. Such symptoms may include but are not limited to cognitive and / or motoric symptoms, progressive nervous system deterioration, problems initiating and controlling muscles and / or movement, early blindness, seizures, and / or spasticity. In embodiments the patient experience or suffer headache, loss of feeling or tingling, weakness or loss of muscle strength, loss of sight or double vision, memory loss, impaired mental ability, lack of coordination, muscle rigidity, tremors and seizures, back pain, muscle wasting, and / or slurred speech. A skilled person in the art is able to diagnose said symptoms without undue effort.

[0169] As used herein, “microgliopathy” comprises any pathological condition of the microglia. Microgliopathies are disorders resulting from the dysfunction of microglia, the specialized immune cells in the central nervous system (CNS) responsible for maintaining CNS health by pruning damaged neurons, clearing debris, producing neuroprotective substances, and fighting infections. Dysfunctional microglia can lead to various neurological and psychiatric disorders. Primary microgliopathies are directly caused by mutations in genes such as TREM2, TYROBP, and CSF1 R, with diseases like Nasu-Hakola disease and adult-onset leukoencephalopathy with axonal spheroids (ALSP) as examples. Secondary microgliopathies occur due to other CNS diseases, including Alzheimer's and Parkinson's disease. ln embodiments, the present invention relates to a pharmaceutical combination for use in the treatment of CNS diseases, preferably microgliopathies. In embodiments, the present invention relates to a pharmaceutical combination for use in the treatment of a microgliopathy, preferably a primary and / or secondary microgliopathy.

[0170] Treatment

[0171] As used herein, the terms “patient,” "individual," and "subject" are often used interchangeably and refer to any animal that exhibits a symptom of a disease, disorder, or condition that can be treated with the pharmaceutical combination and methods disclosed herein. In preferred embodiments, a subject includes any animal that exhibits symptoms of a disease, disorder, or condition associated with the central nervous system, e.g., a neurological condition and / or a mental disorder, including lysosomal storage disease (LSD), Parkinson's disease, Alzheimer's disease, frontotemporal dementia, multiple sclerosis, Rett syndrome, autism spectrum disorder, depression, anxiety disorders, neuropathic pain, and / or schizophrenia, preferably Sandhoff disease or Tay-Sachs disease, that can be treated with methods disclosed herein. Suitable subjects include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates and, preferably, human patients are included.

[0172] As used herein, the terms “(medical) disease,” “(medical) disorder,” and “(medical) condition” are often used interchangeably.

[0173] As used herein, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can optionally involve either the reduction or amelioration of symptoms of the disease or condition or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease, condition, or associated symptoms. The phrase “therapeutically effective” is intended to include, within the scope of sound medical judgment, excessive toxicity, irritation, and / or other problems or complications but commensurate with a reasonable benefit / risk ratio.

[0174] As used herein, "prevent" and similar words such as "prevented," "preventing," or "prophylactic," etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0175] The present invention relates to a pharmaceutical combination comprising a CSF1 R inhibitor and a bone marrow cell population for use in the treatment of central nervous system (CNS) diseases.

[0176] In embodiments, the pharmaceutical combination for use in the treatment of a medical condition of central nervous system (CNS) diseases comprises administering an effective amount, e.g., a therapeutically effective amount of the CSF1 R inhibitor and the bone marrow population contemplated herein. The quantity and frequency of administration will be determined by such factors as the condition of the patient and the type and severity of the patient's disease, although clinical trials may determine appropriate dosages.

[0177] Administration

[0178] According to the present invention, the term “combined administration,” otherwise known as coadministration or joint treatment, encompasses in some embodiments the administration of separate formulations of the compounds described herein, whereby treatment may occur within minutes of each other, in the same hour, on the same day, in the same week or the same month as one another. Alternating administration of two agents is considered as one embodiment of combined administration. Staggered administration is encompassed by the term combined administration, whereby one agent may be administered, followed by the later administration of a second agent, optionally followed by administration of the first agent, again, and so forth. Simultaneous administration of multiple agents is considered to be one embodiment of combined administration.

[0179] A combined therapy or combined administration of one agent may precede or follow treatment with the other agent to be combined by intervals ranging from minutes to months. In embodiments where the second agent and the first agent are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the first and second agents would still be able to exert an advantageously combined synergistic effect on a treatment site. In such instances, it is contemplated that one would contact the subject with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other, with a delay time of only about 12 h being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly; however, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0180] In the meaning of the invention, any form of administration of the multiple agents described herein is encompassed by combined administration, such that a beneficial additional therapeutic effect, preferably a synergistic effect, is achieved through the combined administration of the two components of the pharmaceutical combination.

[0181] Pharmaceutical compositions comprising the pharmaceutical combination for administration to a subject can include, in embodiments, at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional. The person skilled in the art is aware of compositions and formulations suitable for pharmaceutical delivery of the pharmaceutical combination disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

[0182] The pharmaceutical combination can, in embodiments, be combined with pharmaceutically acceptable carrier substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions comprising the pharmaceutical combination, conventional non-toxic pharmaceutically acceptable vehicles can be used, which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutical compositions, whether they are solutions, suspensions, or other like forms, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0183] A pharmaceutical combination comprising an CSF1 R inhibitor, and a bone marrow cell population comprised in a (pharmaceutical) composition can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intraocular, intranasal, intrapulmonary, or transdermal delivery, intramuscular, intraocular, subcutaneous, intravenous, intra-arterial, intraarticular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes.

[0184] In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of pharmaceutical combination may, in embodiments, be administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected condition or one or more symptom(s) thereof, wherein the condition is caused by dysfunctional genes associated with a CNS-based immune response.

[0185] The attending clinician can vary dosage to maintain a desired concentration at a target site (for example, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth.

[0186] The instant disclosure also includes kits, packages, and multi-container units containing the herein- described pharmaceutical combination or pharmaceutical compositions comprising the same and / or means for administering the same for use in the prevention and treatment of conditions described herein and other conditions in human subjects.

[0187] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain, using most routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification indicate the skill level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0188] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. However, the disclosure supports a definition of only alternatives and "and / or." Throughout this application, where relevant, the term "about" indicates that a value includes the inherent variation of error for the device, the method employed to determine the value or the variation among the study subjects.

[0189] FIGURES

[0190] The invention is demonstrated by way of example in the following figures. The figures are to provide a further description of potentially preferred embodiments that enhance the support of one or more non-limiting embodiments of the invention.

[0191] Description of the fi cures:

[0192] Figure 1 : Hexb expression is sufficient to ensure CNS homeostasis: CNS-wide microglia replacement rescues Hexb deficient mice, prevents motor symptoms, and restores CNS homeostasis. (A) Experimental scheme of microglia replacement and the experimental setup. (B) Quantification of cortical IBA1+cells in BLZ945- and vehicle-treated Hexb+ / ~ and Hexb' / ~ mice (n = 3 per group). Two-way ANOVA followed by Sidak test for correcting multiple comparisons was used for statistical testing. (C) Representative immunohistochemical images of microglia (IBA1+) in BLZ945- and vehicle-treated Hexb+ / ~ and Hexb' / ~ mice. (D) Bar graphs highlighting the amount of GFP+ Ly6Cl0blood monocytes in bone marrow transplanted and microglia replaced mice. (E) Kaplan-Meier survival curve of transplanted mice. Log-rank (Mantel-Cox) test. (F) Latency to fall in the rotarod assay for transplanted mice. (G) T rend of bodyweight for transplanted mice. (H) Muscle strength in transplanted mice measured by the grip strength test. (I) FACS-based quantification of the percentage of GFP+microglia indicating the microglia replacement efficiency. (J) Representative immunofluorescence images for Iba1 , GFP, and DAPI for the respective experimental groups are shown. Scale bar 500pm (upper panel) and 25pm (lower panel). (K) Correlation of motor symptoms (mean time on rotarod) and microglia replacement efficiency (%GFP+microglia). Spearman r and two-tailed t-test p values are indicated.

[0193] Figure 2: Translational Potential of Microglia Replacement for Lysosomal Storage Disorders (LSDs). (A) Schematic overview of the experimental setup for culturing primary microglia and assessing p-hexosaminidase (Hex) activity in cell culture supernatants. (B) Quantification of Hex activity in supernatants from wild-type microglia after 4 hours of culture (n = 4). (C) Experimental workflow for treating Hexb~ / ~ neural progenitor cells (NPCs) with conditioned media or recombinant HEXB. (D) Hex activity in Hexb~ / ~ NPCs exposed to conditioned media (CM), heat-inactivated CM (hiCM), unconditioned media (non-CM), or media-only controls (n = 4 per group). (E) Immunocytochemical staining of Hexb+ / ~ NPCs shows co-localization of His-tagged Hex with the lysosomal marker LAMP1 in TuJ1+neurons, indicating successful lysosomal targeting. (F) GM2 ganglioside levels in CM-treated Hexb~ / ~ NPCs (n = 4), demonstrating metabolic correction. (G) Hex activity in Hexb~ / ~ NPCs co-treated with His-tagged recombinant HEXB and inhibitors of endocytosis: EIPA (macropinocytosis inhibitor), Wortmannin (PI3K inhibitor), M6P (blocks M6P receptors), and combined treatment (EIPA + M6P). Sample sizes: rHEXB only (n = 38), EIPA (n = 9), Wortmannin (n = 16), M6P (n = 20), EIPA + M6P (n = 11), untreated (n = 8). (H) Proposed model: In the healthy brain, microglia secrete lysosomal enzymes at high levels. These enzymes are taken up by surrounding neuroectodermal cells via M6P receptor-mediated endocytosis and targeted to lysosomes, where they remain functionally active.

[0194] Figure 3: Ex vivo gene therapy model. (A) Experimental design: Bone marrow from Hexb-deficient donors is transduced with a viral vector carrying a functional Hexb gene. The modified bone marrow, together with helper bone marrow to support engraftment, is transplanted into preconditioned recipients treated with BLZ945 and irradiation. (B) Peripheral chimerism: Flow cytometry analysis demonstrates high levels of donor-derived cells in the peripheral blood of transplanted mice, confirming effective hematopoietic engraftment. (C) Microglial replacement: Flow cytometric quantification of brain-resident myeloid cells reveals efficient microglial replacement by donor- derived cells, indicating that the transduced bone marrow is capable of generating microglia in vivo.

[0195] Figure 4: Hexb expression in the murine brain is highly microglia-enriched throughout CNS conditions, brain regions and development. (A) UMAP of individual microglia from different conditions. 5xFAD and APP23 mice were used as models for Alzheimer’s disease, SOD1 mice for amyotrophic lateral sclerosis, R6 / 2 mice for Huntington’s disease, and cuprizone-treated mice (Cup) to model demyelination. Each dot represents a single cell. Colors correspond to the condition investigated. Specific disease-associated microglia populations are detectable during demyelination and neurodegeneration. (B) Violin plot depicting different microglial core genes and their expression during demyelination and neurodegeneration. (C) Schematic overview of HexbtdTgene locus. A T2A- tdTomato cassette was inserted after exon 14 before the stop codon allowing the expression of tdT and Hexb under the control of the endogenous Hexb gene locus. The self-cleaving peptide T2A ensures the separation of HEXB and tdT proteins. (D) Representative immunofluorescence images of P56 HexbtdT / tdTmice showing high tdT positivity in P2RY12+microglia (yellow) but not NeuN+neurons (green) in the cortex. Triangles point to tdT+microglia. (E) Quantification of tdT+CNS cells. Each symbol represents one individual mouse (n = 4), mean + s.e.m is shown. At least 1000 cells per individual were counted. pvMD : perivascular macrophage, mMD: leptomeningeal macrophage. (F) Quantification of tdT+microglia (IBA1+P2RY12+) in different brain regions at 56 days of age. Symbols represent individual mice (n = 4), mean + s.e.m is shown. At least 1000 cells per individual were counted. (G) Quantification of tdT+cortical microglia (IBA1+, green) at different ages (embyronic (E) day 14.5, postnatal (P) days 1 and 56. Symbols represent individual mice (n = 4) mean + s.e.m is shown. At least 1000 cells per individual were counted.

[0196] Figure 5: Widespread and pronounced early-onset lysosomal activation of microglia is a key feature of Hexb-mediated pathology. (A) Latency to fall in the rotarod assay for Hexb'' (n = 15), Hexb+ / ~ (n = 15) and Hexb+ / +(n = 15) mice. (B) Kaplan-Meier survival curve of Hexbv' (n = 15), Hexb+ / ~ (n = 15) and Hexb+ / +(n = 15) animals. (C) Immunohistochemical pictures of sagittal brain sections from P120 Hexb'' and Hexb+ / ~ mice showing IBA1+microglia (brown). Microglial density (color-based) and APP+deposits (black dots) are indicated (n = 4 per group). (D) Top: Immunofluorescence images of IBA1+microglia (red) highlighting CD68+lysosomes (green) from the thalamus at P120. Bottom: 3D reconstruction of IBA1+microglia (red) and CD68+lysosomes (green). (E) Quantitative analysis of microglial morphologies. At least 3 cells per mouse were measured. (F) Quantification of IBA1+microglia in the thalamus over disease course. (G) Representative immunohistochemical pictures of Mac-3+microglia from the thalamus at P120 (left) and quantification thereof (right). . At least 500 cells per mouse were measured. (H) Immunohistochemical images from the thalamus at P120 (left) and quantification (right) of GFAP+astrocytes . At least 300 cells per mouse were measured. (I) Typical immunohistochemical pictures from the thalamus at P120 (left) and quantification (right) of APP+deposits in the thalamus. At least 300 deposits per mouse were measured. Data shown as mean ± s.e.m. Statistical analyses: oneway ANOVA with Tukey’s post hoc test (A); log-rank test (B); two-tailed Student’s t-test (E); two-way ANOVA with Sidak's test (F-l); each symbol in E-l represents an individual mouse. The color code represents the genotype (orange: Hexb'7", blue: Hexb+ / ~).

[0197] Figure 6: Molecular census of Hexb-deficient mouse brains. (A-C) Uniform manifold approximation and projection (UMAP) visualization of 103,201 individual nuclei from the thalamus of P7 and P120 Hexb'7' and Hexb+ / ~ mice captured by snRNA-seq. (D) Heat map of genes (rows) and dot plots for gene ontology (GO) terms associated with each cluster of microglia shown in c. Key genes are highlighted. Dot plots show selected and enriched GO terms of the respective cluster. Colors in the heat map correspond to normalized scaled expression. Dot color reflects the adjusted p value from a hypergeometric over-representation test with Benjamini-Hochberg correction applied for multiple comparisons. (E) Volcano Plots show the differentially expressed genes between the indicated microglial clusters shown in c. MAST test was used for statistical testing. (F) Heat maps showing the levels (Iog2(fold change)) of lysosome (left) and autophagy (right) pathway-related genes comparing the clusters shown in c. (G) Feature plots depicting most significantly differentially expressed genes in disease clusters.

[0198] Figure 7: Absence of Hexb results in characteristic temporospatial GM2 accumulation which induces microglial production of proinflammatory cytokines via MGL2. (A) Volcano plot indicating the differentially regulated lipids between Hexb'' (n = 6) and Hexb+ / ' (n = 3) mice measured by untargeted lipidomics (liquid chromatography mass spectrometry (LC-MS)) at P120. Two-tailed Welch’s t-test was used for statistical testing. (B) Spatial MALDI mass spectrometry imaging (MALDI-MSI) on Hexb'' and Hexb+' brains at P0 (upper row), P7 (mid row), and P120 (bottom row). For each indicated ganglioside, ion images representative for three biological replicates are shown. Color scale represents a visual map of the intensities (in arbitrary units) of the ion images. (C) MALDI MSI on the thalamus of Hexb'7' and Hexb+' mice at P120. For each indicated ganglioside, ion images representative for three biological replicates are shown. Color scale represents a visual map of the intensities (in arbitrary units) of the ion images. Triangles points to the centromedian (CM) and the parafascicular nucleus (PF). (D) Hierarchical clustering of selected gangliosides in the indicated CNS specimen. Color scale indicates the z-score. (E) Experimental scheme. (F-H) Left: Absolute cytokine and chemokine levels in the supernatant after culturing primary microglia upon overnight ganglioside stimulation. Data are shown as means ± s.e.m. from 4 independent replicates. Two-way ANOVA followed by Sidak's multiple comparison test was used for statistical testing. Right: Log2 fold changes (color scale) are shown relative to the unstimulated condition within each genotype. Statistical significance was assessed using one-way ANOVA with Dunnett’s correction. - Iog10(p) is encoded in color intensity (heatmap), and cells marked with # indicate p < 0.05. F shows a comparison of Hexb'7' and Hexb+7+microglia, G highlights the effect of MGL blockade (MGL Ab vs. isotype control), and H displays responses to different gangliosides (GM1 , GM2, GM3). Figure 8: Joint microglial secretion and neuronal uptake of Hex sustain CNS homeostasis and prevent Sandhoff disease. (A-B) Kaplan-Meier survival curves (A) and rotarod performance (B) of Hexbmi(n = 15), Cx3cr1Cre / +Hexbmi(n = 15), NesCre7+HexbM(n=15), and Cx3cr1Cre / +.NesCre / +.Hexbm(n = 14). (C) Hex activity in whole brain homogenates at indicated times (n = 4). (D-E) Activity in microglia (D) and neurons (E): HexbM(n = 4), Cx3cr1Cre / +-.HexbMI(n = 5 / 6), NesCre / +.Hexbfl / fl(n = 6), Cx3cr1Cre / +.NesCre / +.Hexbm(n = 4), Hexb'' (n = 4). (F) Quantification of HEXB+cortical neurons: Hexbmi(n = 4), Cx3cr1Cre / +-.Hexbmi(n = 4), NesCre / +Hexbf, / fl(n = 4), and Cx3cr1Cre / +.NesCre / +.Hexbfl / fl(n = 3). (G) I BA1+microglia in the thalamus at P245: HexbM(n = 4), Cx3cr1Cre7+.Hexbfl7f!(n = 5), NesCre / +-.HexbMI(n = 4), and Cx3cr1Cre / +NesCre / +Hexbm(n = 4). (H) Hex activity in primary wild-type microglial supernatants after 4 h (n = 4). (I) Same, after Golgicide A pretreatment:0 pM: n = 12, 1 pM: n = 6, 3.5 pM: n = 3, 5 pM: n = 6, 10 pM: n = 9. (J) Experimental scheme. (K) Activity in Hexb'' NPCs treated with conditioned media (CM), heat-inactivated CM (hiCM), unconditioned media (non-CM), or CM-only wells (n = 4 each). (L) Immunocytochemistry of Hexb+' NPCs (TuJ1+) shows lysosomal (LAMP1+) localization of His-tagged Hex. (M) GM2 levels in CM-treated NPCs (n = 4). (N) Activity in Hexb'7' NPCs co-treated with His-tagged Hex and endocytosis inhibitors. rHEXB only: n = 38, EIPA: n = 9, Wortmannin: n = 16, M6P: n = 20, EIPA + M6P: n = 11 , untreated: n = 8. Data shown as mean ± s.e.m. Statistical analyses: log-rank test (A); one-way ANOVA with Tukey’s post hoc test (D-l, K, M, N); two-way ANOVA with Dunnett’s test (C); each symbol in H, I, K, M, N represents a technical replicate.

[0199] Figure 9: Expression of Hexb by bone marrow-derived MLCs rescues lethal CNS phenotype and restores brain homeostasis. (A) Representative immunofluorescence images at P245 showing IBA1 (red), GFP, and DAPI (blue). Triangles indicate IBA1+GFP+replaced microglia. (B-E) Microglia GFP expression by flow cytometry (B), Kaplan-Meier survival analysis (C), rotarod performance (D), and body weight monitoring (E). BLZ + Het -> KO (dark green, n = 12), BLZ + Het - Het (blue, n = 10), BLZ + KO -> KO (red, n = 7), vehicle + KO - Het (grey, n = 10), and KO (orange, n = 7) were analyzed. (F) Correlation between motor function and microglia replacement efficiency. Spearman r and two-tailed t-test p values are indicated. (G) Bar graphs depicting GM2 ganglioside deposition in brain homogenates at P120 (n = 6 per group). (H) Volcano plots of differentially regulated lipids at P120 (n = 6 per group). (I) Hex activity in whole brain homogenates (n = 4 per group) measured at indicated times. (J) Hex activity in neurons at P120 (BLZ + Het -> KO (n = 5), BLZ + KO - KO (n = 4), BLZ + Het - Het (n = 3), and vehicle + KO - Het (n = 4), and KO (n = 3)). (K) Left: HEXB immunohistochemistry in the cortex at P120. Arrows mark neuronal (black) and microglial (green) HEXB+cells. Right: Quantification of HEXB+ neurons (BLZ + Het -> KO (n = 5), BLZ + KO - KO (n = 4), BLZ + Het - Het (n = 3), and vehicle + KO - Het (n = 4). (L) Relative Hexb gene expression and Hex activity in microglia from wildtype mice, Cx3cr1GFP +mice, and microglia-replaced mice, separated by GFP status. Data shown as mean ± s.e.m. Statistical analyses: one-way ANOVA with Tukey’s post hoc test (B-E, H, J-L); log-rank test (C); two-tailed Welch’s t-test (G); two-way ANOVA with Tukey’s post hoc test (I).

[0200] Figure 10: Microglial phenotypes from Sandhoff patients mirror the disease hallmarks observed in Hexb'7' mice. (A) Typical immunohistochemical images thalamic brain sections showing IBA1+(brown) microglia of one postmortem Sandhoff disease patient. Representative hematoxylin and eosin combined with luxol fast blue (H&E-LFB) stain reveals lipid accumulations in thalamic neurons (black arrows) and microglial cells (green arrow). APP immunohistochemistry - T1 - displays extracellular deposits in the Sandhoff disease affected thalamus. Bielschowsky (Biel) stain highlights swollen axons (arrows) Orange color indicates Sandhoff patient, blue unaffected controls. (B) B)-C) UMAP visualization of 14,657 individual nuclei from the thalamus of two Sandhoff disease patients and unaffected controls with in total 1 ,182 immune cells (without ? cells) captured by snRNA-seq. (D) Heat map of genes (rows) and dot plots for gene ontology (GO) terms associated with each cluster of microglia shown in c. Key genes are highlighted. Colors in the heat map correspond to normalized scaled expression. Dot color indicates adjusted p-values from overrepresentation tests with Benjamini-Hochberg correction. (E) Volcano Plots show the differentially expressed genes between the indicated microglial clusters. MAST test was used for statistical testing. (F) Scatter Plot depicting the DEGs in mouse and man with selected genes highlighted. Axes indicate expression changes in mouse (X) and human (Y); genes with adjusted p < 0.05 are shown. (G) MALDI-MSI on cortex and thalamus of a Sandhoff patient and control shows spatial distribution of gangliosides. Ion images reflect signal intensities (arbitrary units).

[0201] Figure 11 : Hexb expression in the murine brain is highly microglia-restricted throughout brain regions and development. (A) Representative immunofluorescence images of P56 HexbtdT7tdTmice showing no tdT positivity in CD206+perivascular and leptomeningeal macrophages (green), SOX9+astrocytes (green) or OLIG2+oligodendrocytes (green) in the cortex. Triangles point to tdT+microglia. pvMD: perivascular macrophage, mMD: leptomeningeal macrophage. (B) Typical immunofluorescence images of tdT+microglia (IBA1+P2RY12+) in different brain regions at 56 days of age. (C) Immunofluorescence images of tdT+cortical microglia (IBA1+, green) at different ages (embyronic (E) day 14.5, postnatal (P) days 1 and 56. (D) Expression of tdT by microglia in HexbtdT7tdTmice measured by flow cytometry. Histograms show the expression levels of tdT at different ages. Hexb+ / +mice are used as controls. Each histogram displays all individual data derived from the indicated number of mice. (E) Immunofluorescence images of P56 HexbtdT tdT.Cx3cr1GFP +mice showing tdT and GFP overlap in IBA1+microglia but not CD206+perivascular or meningeal macrophages. Triangles point to tdT+microglia or tdT' CAMs. (F) Immunofluorescence images of P56 HexbtdT / tdT. Thy1GFP / +mice showing tdT expression only in IBA1+microglia but not GFP+neurons.

[0202] Figure 12: Hexb deficiency leads to microglial activation, astrogliosis, and axonal damage throughout the murine brain with regional differences. (A) Development of body weight over disease course (left) and strength in the Grip Strength Test (right) for Hexb'' (n = 15), Hexb+ / ' (n = 15), and Hexb+ / +(n = 15) controls. (B) Measurement of blood marker for liver and kidney damage in Hexb+ / ' and Hexb'' mice. One symbol indicates one biological replicate. (C) TNF and IL-6 levels in the blood of Hexb+ / _and Hexb'7' mice measured by ELISA. Paired t-test comparison was used for statistical testing. (D) Quantification of APP (axonal damage), GFAP (astrocytosis), IBA1 (microgliosis), and Mac-3 (lysosomal microglia activation) in different brain regions (cortex, cerebellum grey and white matter, hippocampus, thalamus, and pons / medulla) at different time points (P0, P7, P28, P56, P85, P120) for Hexb'7' (n = 4) and Hexb+' (n = 4) controls. (E) Representative immunohistochemical image of IBA1 / Mac-3 double-positive cells in the thalamus of P120 Hexb'7' mice. (F) Representative immunofluorescence images indicating lysosomal activation in P7 Hexb'7' (orange) and Hexb+' (blue) microglia. (G) Left: Representative immunohistochemical images of P2RY12+and TMEM119+cells in the thalamus at P120Right: Quantification Each dot represents one individual mouse. Th: thalamus, Cwm: Cerebellum - white matter, P / M: pons / medulla, Cgm: Cerebellum - grey matter, Ctx: cortex, H: hippocampus. Data shown as mean ± s.e.m. Statistical analyses: Two-tailed Student’s t-test was used for statistical testing (B-C); Two-way ANOVA followed by Sidak's multiple comparison test was used for statistical testing (D,G).

[0203] Figure 13: snRNA-seq reveals CNS cell-type composition and microglial heterogeneity across disease states. (A) Selected marker genes associated with each cell type highlighted in Figure 6a. (B) Typical marker genes associated with each immune cell type highlighted in Figure 6b. (C) Dotplot depicting common microglial homeostatic and disease-associated genes among clusters shown in Figure 6c. (D) Heat map featuring the top cell-type-specific marker genes across the major cell types. The color bar indicates gene expression. (E) Marimekko plot depicting the different cell type compositions separated by age or genotype. (F) UMAP and Marimekko chart of microglia and CAMs depicting the proportions of Hexb'' and Hexb+ / ' microglia for each cluster. (G) UMAP visualization of microglia cluster from different conditions shown in Figure 4a. Here, microglia from P120 Hexb'' microglia are integrated. (H) Marimekko plot depicting the cluster proportions for the indicated conditions. (I) Heat map featuring the top cluster marker genes. Shared Hexb'' disease genes are highlighted. (J) UMAP visualization of microglia shown in g. Color code indicates their belonging to the homeostatic or disease condition. (K) UMAP visualization of microglia shown in g. Color code indicates the respective condition.

[0204] Figure 14: Analysis of ganglioside storage in a temporospatial manner. (A) Bar graphs depicting dysregulated ganglioside deposition in brain homogenates of Hexb'7' (n = 6) and Hexb+' mice (n = 3) at 120 days of age measured by untargeted lipidomics (LC-MS). (B) Spatial MALDI mass spectrometry imaging (MALDI-MSI) on Hexb'7' and Hexb+' brains at P0 (upper row), P7 (mid row), and P120 (bottom row). For each indicated ganglioside, ion images representative for three biological replicates are shown. Color scale represents a visual map of the intensities (in arbitrary units) of the ion images. (C) Heatmap showing all differentially regulated and annotated lipids in the brain of PO, P7, and P120 Hexb'7' (n = 3) and Hexb+ +(n = 3) control mice. Color scale indicates the z-score. (D) MALDI MSI on the thalamus of Hexb'7' and Hexb+' mice at P120. For each indicated ganglioside, ion images representative for three biological replicates are shown. Color scale represents a visual map of the intensities (in arbitrary units) of the ion images. (E) Representative immunofluorescence images of GM2 storage in neurons and microglia in Hexb'7' mice. Triangles point to GM2+IBA1+microglia or GM2+NeuN+neurons. (F) Representative immunofluorescence images of GM2 storage (green) in lysosomes (LAMP1+, yellow) in microglia (IBA1+, red, top panel) or neurons (NeuN+, red, bottom panel), respectively. Orange color indicates Hexb'7' mice and blue color Hexb+' mice. (G) Quantification of lysosomal GM2, shown as the integrated fluorescence intensity of GM2 signal within LAMP1+lysosomal areas, normalized to the cell area defined by IBA1+(microglia) and NeuN+(neurons) signals, respectively. Each symbol indicates one mouse. (H) Data shown as mean ± s.e.m. Statistical analyses: Unpaired Student’s t-test comparisons were used for statistical testing (A); two-way ANOVA followed by Tukey’s test for correcting multiple comparisons was used for statistical testing (G).

[0205] Figure 15: In vitro and in vivo microglial responses to gangliosides and cortical dysfunction in Hexb-deficient mice. (A-B) Cytokines and chemokine concentrations in supernatants from primary wildtype microglia cultured with the indicated amount of GM2, with / without EGTA or GalNAc. Data from 4 technical replicates. (C) Fold changes of indicated cytokines normalized to the unstimulated condition (based on A and B). (D) Schematic of in vivo experimental design. (E-F) Cytokines / chemokine levels in brain lysates following ICV antibody injections. (G) Quantitative PCR analysis of indicated target genes. (H) Experimental setup. (I) Representative traces (top) and current-clamp recording protocol (bottom). Note fewer action potentials (APs) and absence of voltage sag (arrow) in Hexb'7' compared to Hexb+ / ~ mice. (J) No difference in basic neuronal attributes between the genotypes (Hexb+ / ". 4 animals, 11 neurons; Hexb'' 4 animals, 11 neurons). (K) Hexb'' mice display reduced AP firing in response to depolarizing current injections across the entire range tested (left), along with an increase in rheobase, the amount of current required to elicit an AP (right, Hexb+ / '-’. 4 animals, 11 neurons; Hexb'7". 4 animals, 11 neurons). (L) Representative traces of individual APs. (M) Hexb'' mice show increased AP halfwidth, whereas AP amplitude and threshold are similar between the genotypes (Hexb+ / ". 4 animals, 10 neurons; Hexb'"A animals, 10 / 8 / 8 neurons respectively). (N) Voltage sag during hyperpolarization (arrow in panel b) is reduced in Hexb'7' mice (Hexb+ / ". 4 animals, 11 neurons; Hexb'7"A animals, 11 neurons). (O) Voltage-clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs) show, robust reduction of sEPSC frequency in Hexb'7' mice. sEPSC amplitude is similar in both genotypes (right, Hexb+'-. 4 animals, 9 neurons; Hexb'7' A animals, 8 neurons). Data shown as mean ± s.e.m. Statistical analyses: Two-way ANOVA followed by Sidak's multiple comparison test (A-B.E-G); One-way ANOVA followed by Dunnett’s test (C); two-tailed Student’s t-test (l-K, M-O); two tailed Mann- Whitney (K). For l-O, dots correspond to individual neurons.

[0206] Figure 16: Microglial and neuronal Hexb jointly drive Sandhoff disease pathogenesis. (A) Schematic overview of genetic targeting. Hexb™1were obtained by crossing the B6.Hexbtm1a(EUCOMM)Hm9U / H line with a FLP deleter. Newly generated Hexb™1mice with LoxP sites (black triangles) around exon 2 were crossed to Cx3cr1Cre +and NesCre +mice. (B)-C) Relative Hexb gene expression in FACS-sorted microglia (B) and bead-purified neurons (C) among different genotypes measured by qPCR. (D) Relative Syt1 (neuronal marker gene), Gfap (astocytic marker gene), Itgam (microglia marker gene), and Plp1 (oligondendroglial marker gene) gene expression in bead-purified neurons among different genotypes measured by qPCR. (E-F)Development of bodyweight (E) and muscle strength (F) for Hexb™1(n = 15), Cx3cr1Cre7+.Hexbflfl(n = 15), NesCre / +.Hexb™1(n=15), and Cx3cr1Cre +.NesCre +-.Hexb™1(n = 14) mice. (G) Representative immunohistochemical images of brain sections from the indicated genotypes. Top row: Microglia immunostained for P2RY12 (red), HEXB (brown), and counterstained with hematoxylin (Htx, blue). Bottom row: Neurons stained for NeuN (red), HEXB (brown), and Htx (blue). Insets show higher magnification views with yellow lines indicating the paths used for intensity profile quantification below. Triangles highlight intracellular HEXB-double positive structures. Graphs display greyscale intensity profiles of the deconvoluted staining signals along the yellow lines (red = P2RY12 or NeuN, brown = HEXB, blue = Htx). Data shown as mean ± s.e.m. Statistical analyses: one-way ANOVA with Tukey’s post hoc test (E-F).

[0207] Figure 17: Microglial Hex secretion and uptake by other cell types. (A) Experimental scheme of microglia culture and Hex activity measurement in cell culture supernatants. (B) Immunoblot of culture supernatant and exosome isolates for TSG101 (exosomal marker). (C) Diagram of major protein secretion pathways and their molecular inhibitors. ER: endoplasmic reticulum, EGTA: ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, MVB: multivesicular body. (D)-F) Hex activity in microglial supernatants treated with indicated concentrations of Brefeldin A (D), Vacuolin-1 and / or lonomycin (E), or EGTA, Thapsigargin, and BAPTA-AM (F). (G) Enzyme activity in conditioned media (CM), heat-inactivated CM (hiCM), and unconditioned media (non-CM). (H) Hex activity in lysates of Hexb+ / ~ NPCs treated with CM, hiCM, non-CM, or CM added to wells without cells ("no cells") for 24h. (I) Top: Experimental setup using transwell inserts. Bottom: Hsc, activity in lysates of Hexb'' and Hexb+ / ~ NPCs after co-culture with Hexb+ / +microglia ± Brefeldin A (BFA). (J) Immunoblots of NPC lysates after 6h treatment with recombinant Hex-His. (K) GM2 levels in NPC lysates after CM treatment. (L) Experimental scheme for primary Hexb'' fibroblast culture. (M) Immunoblots of fibroblast lysates after 6h treatment with recombinant Hex-His. (N-O) Enzyme activity in fibroblast lysates ± recombinant enzyme and following pretreatment with mannose-6-phosphate (M6P), M6P receptor (M6PR) antibody, wortmannin, or EIPA. (P) Immunocytochemistry if His- tagged enzyme in Hexb'7' fibroblasts (vimentin+, His+). Triangles point to intracellular His+inclusions. (Q) Schematic of chimeric organotypic hippocampal slice cultures (OHSCs). (R) Hex activity in OHSC supernatants, exosomes, and exosome-depleted fractions from Hexb+ / ' and Hexb'' slices ± clodronate treatment and microglia transplantation. (S) Correlation of enzyme activity with microglia density. (Spearman r, p values from two-tailed t-test). (T) Immunohistochemistry for NeuN (red) and HEXB (brown) counterstained with hematoxylin (Htx) in microglia-transplanted Hexb'7' slices at d17. Graphs display greyscale intensity profiles. (U) Immunohistochemistry and quantification of IBA1+microglia in OHSCs at d17. Data shown as mean ± s.e.m. Statistical analyses: one-way ANOVA with Tukey’s post hoc test (D-I,K,O); two-way ANOVA with Sidak’s test (O,R).

[0208] Figure 18: Microglial Hexb expression ensures CNS homeostasis. (A) Experimental scheme for microglia replacement. WBI: whole body irradiation. BMT: bone marrow transplantation. (B-C) Quantification and immunohistochemistry of cortical IBA1+cells in BLZ945- and vehicle-treated Hexb+' and Hexb'7' mice (n = 3 per group). (D) FACS analysis of GFP+Ly6Cl0blood monocytes in the transplanted and microglia replaced mice measured by FACS. (E) Quantification of %GFP+IBA1+parenchymal cells in the cortex and thalamus (replacement efficiency) from Figure 9a. (F) Grip strength assessment in transplanted mice. (G) Ganglioside levels in brain homogenates of transplanted Hexb'7' (n = 6) and Hexb+' animals, and untreated Hexb'7' controls (n = 6). (H-l) Bar graph highlighting the amount of GFP+Ly6Cl0blood monocytes related to Figure 6I (H) and of neonatally transplanted and microglia replaced mice (I). (J) Flow cytometry of GFP expression in microglia of neonatally transplanted mice. (K-L) Kaplan-Meier survival curve (K) and rotarod performance (L) of neonatally transplanted mice. BLZ + Het -> KO (n = 12), BLZ + Het -> Het (n = 10), and neonatally BLZ + Het -> KO (n = 4). (M-O) UMAP of 44,957 individual nuclei from the thalamus of transplanted and untransplanted Hexb'7' and Hexb+' mice captured by snRNA-seq. (P- Q) Marker genes identifying cell types and immune populations. (R) Dotplot of homeostatic and disease-associated microglial genes by clusters. (S) Heat map of key cluster-specific microglial genes. (T) Volcano plot depicting DEGs between cO and c1 in transplanted / 7exb' / _mice. (U) Marimekko chart of microglia depicting the proportions of transplanted and untransplanted Hexb'7' and Hexb+' microglia for each cluster. (V) Violin plot highlighting the expression of core microglial genes in transplanted and untransplanted Hexb'7' and Hexb+' microglia (c1).

[0209] Data shown as mean ± s.e.m. Statistical analyses: two-way ANOVA followed by Sidak’s test (B); one-way ANOVA with Tukey’s post hoc test (E,G,L).

[0210] Figure 19: Histological, transcriptional, and lipid changes in Sandhoff disease brains. (A) Quantification of I BA1+microglia in postmortem brain tissue of Sandhoff disease patients and unaffected controls. Orange indicates Sandhoff disease, blue unaffected controls. Th: thalamus, Cwm: Cerebellum - white matter, Cgm: Cerebellum - grey matter, Ctx: cortex, WM: subcortical white matter. Each bar represents one patient. (B) Immuohistochemistry for phagocytic and lysosomal markers: KIM1 P, p22phox, lysozyme, and LAMP2. (C) H&E staining thalamic sections of Sandhoff disease patients and healthy controls. (D) H&E stains depicting cellular ganglioside deposition and an enlarged hypercellular perivascular space. (E) Bielschowsky (Biel) stain highlighting the axonal network. (F) Immunostaining for axonal- and neurofilament-associated proteins: SMI31 (phosphorylated neurofilaments), SMI35 (non-phosphorylated neurofilaments), and SMI312 (pan-axonalhighly phosphorlylated neurofilaments marker). Note the abnormal accumulation of SMI31 in perikarya of degenerationg neurons with ganglioside accumulation as well as axonal swellings. (G) Heat map featuring the top cell-type-specific marker genes across the different cell types. (H) Marimekko charts depicting the proportions of each cell type or cluster separated by disease condition. (I) Feature Plots depicting cluster defining genes. (J) MALDI MSI of cortex and thalamus from Sandhoff disease patients and unaffected controls. Ion images show spatial distribution of gangliosides; color scale indicates intensity (arbitrary units). (K) Volcano plot indicating the differentially regulated lipids between Sandhoff disease patients and unaffected controls measured by untargeted lipidomics (liquid chromatography mass spectrometry (LC-MS)). Two-tailed Welch’s t-test was used for statistical testing.

[0211] Figure 20: Graphical abstract of experimental findings. Left panel (Homeostasis): In the healthy brain, microglia expressing high levels of Hexb secrete functional Hex, which is taken up by neurons and delivered to their lysosomes.

[0212] Middle panel (Sandhoff disease / model): In the absence of Hexb, both microglia and neurons accumulate GM2 ganglioside in their lysosomes. Neuronal cell death leads to extracellular GM2 release, which is sensed by microglia via the receptor MGL2, recognizing terminal GalNAc residues. This triggers a pro-inflammatory response, including the upregulation and secretion of TNF, IL-6, CCL3, CCL4, and CCL17.

[0213] Right panel (Microglia replacement therapy): Transplantation of wild-type microglia into Hexb- deficient brains restores enzymatic function in neurons via microglial enzyme supply. The replaced microglia secrete functional Hex, thereby reducing neuronal and CNS-wide GM2 accumulation and promoting restoration of CNS homeostasis.

[0214] EXAMPLES

[0215] The invention is demonstrated by way of the examples disclosed below. The examples provide technical support for and a more detailed description of potentially preferred, non-limiting embodiments of the invention.

[0216] Summary of the Examples

[0217] In order to demonstrate the functionality and beneficial properties of the pharmaceutical combination described herein, the following examples are to be considered: BLZ945 treatment

[0218] Bone marrow transplantation and microglia replacement

[0219] Survival analysis

[0220] Behavioral testing

[0221] Translational Potential of Microglia Replacement for Lysosomal Storage Disorders (LSDs)

[0222] Ex vivo gene therapy model

[0223] Following mice were used in the examples: Hexb'' mice (B6.129S-Hexbtm1 Rlp / J) were purchased from Jackson Laboratories (#002914) and backcrossed for five generations with C57BL / 6J mice. In addition, Neu3'':Hexa'' mice were used. Neu3'' animals were received from the CARD center, University of Kumamoto (Japan), Hexa'7' animals were purchased from Jackson Laboratories (#002367) and mated together to generate double knockout animals. Cx3cr1GFP(B6.129P2(Cg)- Cx3cr1fm7 / -,w / J) mice served as bone marrow donors. All mice were bred in-house under pathogen- free conditions. Diseased mice received wet food placed on the cage ground. All animal experiments were approved by the local administration (Regierungsprasidium Freiburg, approval numbers G- 17 / 34, G-21 / 020, and G-22 / 035) and were performed in accordance with the respective national, federal, and institutional regulations.

[0224] Example 1: BLZ945 treatment

[0225] BLZ945 hydrochloride (HY-12768A, MedChemExpress) was dissolved in 20% (2-hydroxypropyl)-P- cyclodextrin (H107, Sigma-Aldrich). A dose of 200 mg per kg bodyweight was applied by oral gavage for seven consecutive days.

[0226] Example 2: Bone marrow transplantation and microglia replacement

[0227] To deplete endogenous microglia, mice received BLZ945 for seven consecutive days. On the day of the transplantation, the last dose was applied. In parallel, mice were treated with neomycin (1.1 g / L; N6386, Sigma) acid water (pH 2.5) to reduce the risk of infection. Recipient mice were lethally irradiated with 9 Gray (Gy) using an RS2000 X-ray irradiator (Rad Source Technologies). Cx3cr1GFPmice served as bone marrow donors. Bone marrow was isolated from the tibias and femurs by flushing with PBS. After red blood cell removal, cells were washed, counted, and resuspended in an appropriate volume of PBS (1 x 107cells per 100 pl). Within two hours after irradiation, mice received donor bone marrow (1 x 107cells) via tail vein injection. Following the injection, treatment with neomycin acid water was continued for another two weeks. Four weeks after transplantation, mice were subjected to blood withdrawal from the facial vein to control for proper reconstitution with donor-derived peripheral blood cells.

[0228] Example 3: Survival analysis

[0229] Upon weight loss (> 10% loss) or occurrence of an impaired righting reflex (> 5 sec), diseased mice were euthanized, and the age was recorded.

[0230] Example 4: Behavioral testing Rotarod Motor coordination was assessed using the rotarod assay. The assay was conducted using a Rota-Rod (Model 47650, Ugo Basile) with accelerating speed (accelerated from 3 to 40 r.p.m. over 300 s). The mice were trained on the accelerating rotarod one day before the first recorded testing. The latency to fall off the rotarod was recorded three times a week until the end of the observation period. Only one trial was conducted per day. A full passive rotation or falling off the rotarod was considered a failure and recorded as “latency to fall”. Grip strength test Muscular strength was assessed with the grip strength test using a Grip-Strength-Meter (Mains). Mice were grasped at their tail and placed on a slight oblique grid with all four limbs. Now, the tail was gently and continuously pulled backward. The maximum force was automatically recorded when the mouse lost its grip. Each mouse was tested three times; the best trial was recorded.

[0231] Example 5: Translational Potential of Microglia Replacement for Lysosomal Storage Disorders (LSDs)

[0232] Lysosomal storage disorders (LSDs) represent a group of over 70 monogenic diseases, most of which follow an autosomal recessive inheritance pattern. While individually rare, they collectively affect approximately 1 in 5,000 individuals. The underlying mutations typically affect genes encoding lysosomal hydrolases, membrane proteins, transporters for lipids and ions, as well as enzyme modifiers or activators. Clinically, LSDs are heterogeneous; however, many share a prominent pediatric neurodegenerative component, which significantly impacts disease progression and prognosis.

[0233] The rationale for extending microglial replacement therapy to a broader spectrum of LSDs stems from our findings in Hexb-deficient models. It is demonstrated that microglia constitutively secrete p- hexosaminidase (Hex) enzymes, which are taken up by neighboring neurons. Once internalized via mannose-6-phosphate (M6P) receptor- mediated endocytosis, the enzyme is trafficked to lysosomes, where it remains functionally active.

[0234] This mechanism - cross-correction via M6P-tagged lysosomal enzymes - is applicable to other LSDs. Microglia, as the brain’s resident phagocytes, express high levels of various lysosomal enzymes, many of which are secreted. Because all lysosomal hydrolases bear M6P tags, it is likely that these secreted enzymes can be similarly taken up by surrounding neuroectodermal cells, thereby restoring enzymatic activity in deficient cells.

[0235] Consequently, microglial replacement constitutes a promising, broadly applicable therapeutic strategy for the treatment of neurodegenerative forms of LSDs.

[0236] Example 6: Microglia-neuron crosstalk via Hex-GM2-MGL2 maintains brain homeostasis

[0237] RESULTS

[0238] Hexb is a stable microglia gene

[0239] Recent studies have sought to identify microglial genes, which robustly separate them from CAMs or other brain resident cells, aiming to target these cells specifically. Key genes include P2ry12, Tmeml 19, SalH , and Hexb. To identify microglial genes that are robustly expressed during pathology single-nucleus 3’ mRNA sequencing (snRNA-seq) of microglia in five models of neurodegeneration or demyelination was performed. As expected, the several context-dependent microglial clusters emerged (Figure 4a). Among core microglial genes Hexb, P2ry12 and Cx3cr1 showed consistently high expression, whereas Tmeml 19, SalH , Gpr34, Siglech, Olfml3 and Feris were either low or variable (Figure 4b).

[0240] To examine Hexb regulation in health, the HexbtdTreporter line was used (Figure 4c). Of note, virtually all cortical microglia expressed tdTomato (99.06 ± 0.10 %), unlike perivascular macrophages (pvMcp; 9.10 ± 0.89 %), leptomeningeal macrophages (mMcp; 5.04 ± 0.99%), neurons (0.01 ± 0.01 %), astrocytes (0.03 ± 0.03 %) or oligodendrocytes (0 %) (Figures 4d-e, Extended Data Figure 1a). Moreover, microglia were tdTomato+across brain regions and kept Hexb expression over development (Figures 4f,g, Extended Data Figure 11 b-d). Crossbreeding of HexbtdTmice with Cx3cr1GFPor Thy1GFPconfirmed no overlap of Hexb-expressing microglia with GFP+CAMs or neurons, respectively (Extended Data Figure 11 e,f). Prior reports of low Hexb mRNA in murine neurons were confirmed in adult wild type mice (data not shown). In sum, Hexb is a highly stable microglial gene throughout the murine CNS during development, homeostasis and disease.

[0241] Early and robust microglial activation

[0242] Having proven that microglia expressed Hexb at high levels in the murine brain, next its functional role was examined. Hexb knockout (Hexb'7') mice — previously generated as Sandhoff disease model — developed rapidly progressing ataxia and weight loss, with preserved grip strength, and died at postnatal day (P) 114 ± 12 (Figures 5a, b, Extended Data Figure 12a). Notably, no peripheral inflammatory causes of an encephalopathy causing motor symptoms were found in the blood (Extended Data Figures 12b, c), and brains lacked lymphocytic infiltrates (data not shown). In contrast, microglia revealed striking changes in number and morphology (Figures 5c-f, Extended Data Figure 12d). Microglial cell numbers increased by P28 and peaked at P85, indicating early involvement before clinical onset. Densities varied regionally, with the highest counts in thalamus, pons / medulla, and cerebellar white matter (Figure 5c). 3D reconstruction of IBA1+microglia and CD68+lysosomes revealed enlarged lysosomal volumes, fewer terminal / branch points, and shortened processes (Figures 5d,e) Moreover, microglia upregulated the lysosomal activation marker Mac-3 as early as P7 (Figure 5g, Extended Data Figures 12d-f) and downregulated TMEM119 and P2RY12, highlighting their activated phenotype. P2RY12 loss was most evident in the thalamus (51.29 ± 4.23 % of IBA1+microglia) (Extended Data Figure 12g). Astrogliosis only appeared at P85, when mice were already affected clinically (Figure 5h, Extended Data Figure 12d), while APP+extracellular deposits, a sign of axonal damage, emerged late, especially in the thalamus (Figures 5c, j, Extended Data Figure 12d). In sum, lack of Hexb drives early and excessive microglial activation reflected by drastic numeric, morphological and lysosomal changes, while astrogliosis and axonal injury are late events.

[0243] Molecular census of Hexb'' CNS cells

[0244] To explore the molecular basis of microglial activation in Hexb'7' mice, snRNAseq was performed on thalamic nuclei at P7 and P120. Heterozygous mice (Hexb+7‘) served as controls, as they showed no expression differences compared to Hexb+7+microglia. The thalamus was chosen for transcriptomic profiling due to the pronounced microgliosis, severe axonal damage and its known involvement in Sandhoff disease in humans. After quality control, 103,201 nuclei were analyzed, with cell types assigned using the Azimuth tool based on reference gene sets (Figures 6a, Extended Data Figures 13a, b). Immune cell proportions increased notably in aged Hexb'7' mice (Figures 6b, Extended Data Figures 13c, e). Within the immune population, 7 distinct microglia clusters were identified. Clusters cO and 1 comprised virtually all adult control microglia thus representing ‘homeostatic’ clusters. Cluster c2-4 were overrepresented in diseased knockouts, while neonatal microglia (c5) or CAMs lacked disease-associated clusters suggesting a pure microglial involvement in disease (Figures 6c, Extended Data Figure 13d,f). Pseudotime analysis identified a trajectory from transitional cluster c2 to terminal disease cluster c3 and c4 (Figure 6c). These clusters showed reduced homeostatic markers (P2ry12, Cx3cr1 , Gpr34, SalH and Siglech) and increased activation genes (Apoe, Ctsb, Lyst, Csf1 , Tyrobp, B2m, Spp1 , or Cybb) (Extended Data Figure 13d). Clusters c2 and c3 shared upregulation of I gf 1 , Apobed , and Fit 1 (Figures 6d-e, g). Gene ontology analysis linked c2 to cytokine-related terms, c3 to autophagy / phagocytosis pathways, and c4 to interferon responses (Figures 6d, f Compared to other neurodegeneration models (Figure 4a), Hexb-deficient microglia shared general patterns but aligned most closely with clusters 11 and 12 from 5xFAD and APP23 mice. (Extended Data Figures 13g-k). Together, loss of Hexb induces a progressive microglial activation trajectory toward a disease-specific, highly dysfunctional state characterized by altered immune, autophagic, and phagocytic profiles.

[0245] To assess the impact of Hexb deficiency on CNS lipid composition, untargeted lipidomics were performed on cortical homogenates from Hexb'7' and Hexb+7' mice. Among 5825 identified lipids, 196 were significantly upregulated and 59 downregulated in Hexb'7' brains (Figure 7a). Importantly, multiple GM2 species were markedly increased, whereas their Hex-derived products, GM3 gangliosides, were strongly decreased (Extended Data Figure 14a). To map spatial ganglioside distribution, untargeted MALDI-mass spectrometry imaging (MSI) was applied to brain sections at multiple time points. Several physiologically regulated lipid species varied with age [e.g., cardiolipins, sulfatides (SM4), gangliosides (GT 1 , GR3, GD1 , GD3)] but did not differ between genotypes (Extended Data Figure 14c). In contrast, GM2 accumulation was already detectable at PO in knockout brains (Figures 7b, d, Extended Data Figure 14b). Notably, GM2 molecules with shorter fatty acyl chains (e.g. d34: 1 ) predominantly accumulated in neonates, while GM2 molecules with longer fatty acyl chains (e.g. 38:2) were enriched at P120 (Figures 7b, d). This shift was especially pronounced in the thalamus, which showed prominent GM2 accumulation at P120 (Figure 7c, Extended Data Figure 14d). Within the thalamus, GM2 localized to specific nuclei — the centromedian (CM) and parafascicular (PF) — key sources of thalamostriatal projections involved in motor control (Figure 7c). Beyond GM2, pathologically elevated lipids included GA2, asialo-GM2, and BMP 44:12. Immunofluorescence imaging confirmed GM2 storage in neurons and microglia, but not astrocytes and oligodendrocytes (Extended Data Figure 14e). Both microglia and neurons exhibited substantial lysosomal ganglioside burden, with neurons tending towards higher levels (Extended Data Figure 14f,g). Altogether, the u spatial ganglioside map revealed a distinct diseases- associated lipid profile driven by Hexb loss with regionally enriched GM2 accumulation, especially in the thalamus.

[0246] GM2 activates microcilia by MGL2 engagement

[0247] Having identified GM2 as the most dysregulated lipid in Hexb'7' brains and its spatial overlap with microgliosis, its direct effects on microglia were examined. Primary microglia from Hexb'7' and Hexb+ / +control mice were plated into GM2-coated 96-well plates, and cytokine and chemokine secretion was measured (Figure 7e). GM2 induced a dose-dependent release of IL-6, TNF, CCL3, CCL4 and CCL17, with higher levels in Hexb'7' cells, likely due to pre-activation from chronic ganglioside exposure (Figure 7f). To dissect the mechanism, it was focused on macrophage galactose-type lectin (MGL)2, a C-type lectin receptor expressed on dendritic cells, macrophages and microglia that specifically binds to terminal GalNAc present on GM2. While MGL2 is known for endocytosis, it was tested whether it also mediates GM2-driven microglial activation. Indeed, microglia pre-treated with MGL2-blocking antibody no longer responded to GM2 with cytokine release, whereas isotype-treated cells did (Figure 7g). To rule out nonspecific antibody effects, EGTA was used to chelate extracellular calcium, essential for C-type lectin function, or with GalNAc to competitively inhibit MGL2 binding, both of which suppressed cytokine responses, confirming the specificity of MGL2-GM2 signaling (Extended Data Figures 15a-c). Furthermore, GM1 and GM3 — lacking an accessible or present GalNAc — did not induce cytokines (Figure 7h).

[0248] To validate the findings in vivo, MGL2-blocking antibody were administered via ICV injections to Hexb'7' and Hexb+ / ' mice from P10 for three weeks (Extended Data Figure 15d). Injected Hexb'7' brains showed a significant reduction in TNF and CCL4, along with decreased IL-1 a and CXCL9 levels (Extended Data Figure 15f). FACS-purified microglia had decreased Ccl5 and Cx3cl1 mRNA and trends toward reduced 111 b and 1118 mRNA expression (Extended Data Figure 15g). Together, these findings demonstrate that GM2 acts as a specific microglial activator via MGL2, both in vitro and in vivo.

[0249] Hexb loss impairs neuronal excitability

[0250] Having defined the mechanism of GM2-induced microglial activation, next it was investigated weather Hexb deficiency and subsequent GM2 accumulation impair neuronal signaling. Thus, electrical recordings from motor cortex layer 2 / 3 pyramidal neurons were performed in acute slices (Extended Data Figure 15h,i). While input resistance and membrane potential were unchanged (Extended Data Figure 15j), Hexb'7' neurons fired significantly fewer action potentials during depolarizing current steps, indicating reduced excitability (Extended Data Figure 15i,k). In addition, changes in action potential halfwidth (Extended Data Figure 151, m) and voltage sag during hyperpolarization (Extended Data Figure 15n) suggested dysregulation of the underlying conductance. At the network level, Hexb'7' neurons showed reduced frequency of synaptic inputs, reflecting impaired glutamatergic connectivity (Extended Data Figure 15o). Collectively, these data reveal robust deficits in neuron-autonomous and circuit function upon Hexb deficiency and GM2 accumulation.

[0251] Microc / lial & neuronal Hexb drive disease

[0252] Until now, limited cell type-specific targeting tools hindered identification of disease-driving cell types Sandhoff disease. Although loss of neuronal Hexb Hex activity is believed to drive GM2 accumulation and pathology, the high expression of Hexb in microglia (Figures 4c-g) suggests an additional role in disease progression. To dissect cell-specific contributions, exon 2 of the Hexb gene was flanked by loxP sites and generated Hexbfl7flmice (Extended Data Figure 16a). These were crossbred with NesCre7+(targeting neuroectodermal cells, including neurons) and Cx3cr1Cre7+(targeting myeloid cells, including microglia). Each line showed efficient Hexb deletion (Extended Data Figures 16b-d). Surprisingly, neither NesCre / +:Hexbfl / flnor Cx3cr1Cre / +:Hexbfl / flmice recapitulated the phenotype observed in constitutive Hexb'7' mice and survival, motor function, and body weight remained normal (Figures 8a, b, Extended Data Figures 6e, f). In both single knockouts, total brain Hex activity was only slightly reduced (Figure 8c), and neurons retained enzymatic activity and HEXB-positive structures, pointing to a redundant role of microglial or neuronal Hexb expression (Figures 8d-f, Extended Data Figure 16g). Strikingly, only double knockout Cx3cr1Cre / +:NesCre / +:Hexbfl / flmice recapitulated the disease with late-onset motor symptoms, weight loss and premature death (Figures 8a, b, Extended Data Figure 16e). These mice showed drastic reduction of Hex activity and massive loss of HEXB+neurons (Figures 8c, e, f, Extended Data Figure 16g). Microglia also exhibited abnormal morphology and increased cell numbers (Figure 8g). Overall, only a combined deficiency of Hexb in the neuroectodermal and myeloid compartments is sufficient to induce Sandhoff disease.

[0253] Microglia aid neuronal lysosomal function

[0254] Since neither microglia nor neurons alone induce disease, a compensatory mechanism for GM2 turnover involving microglial enzyme release was hypothesized. To test this, Hexb+ / +microglia were cultured and Hex activity was measured in the supernatant (Extended Data Figure 17a). Activity was significantly higher than in medium-only controls and localized to the exosome-depleted fraction, indicating secretion of free Hex (Figure 8h, Extended Data Figure 17b). To determine the secretion pathway, microglia were treated with various inhibitors (Figure 8i, Extended Data Figures 17c-f). Among them, only Golgicide A and Brefeldin A significantly and dose-dependently blocked microglial Hex secretion (Figure 8i, Extended Data Figure 17d) suggesting release via the classical secretory pathway. Thapsigargin and BAPTA-AM also reduced enzyme secretion, implicating intracellular Ca2+in sustained secretion, while vacuolin-1 and EGTA had no effect (Extended Data Figure 17f).

[0255] To assess neuronal uptake, Hexb'7' and Hexb+7' neural progenitor cells (NPCs) were treated with His- tagged recombinant Hex or conditioned media (CM) from primary wild-type microglia (Figure 8j, Extended Data Figure 17g). CM induced a time-dependent increase in enzymatic activity in NPCs (Figure 8k, Extended Data Figure 17h), corroborated by transwell co-culture experiments, confirming enzyme transfer is independent of direct cell contact (Extended Data Figure 17i). Western blotting confirmed Hex uptake by NPCs (Extended Data Figure 7j), and immunofluorescence imaging validated lysosomal localization of internalized enzyme (Figure 8I). Notably, CM-treated Hexb'7' neurons stored less GM2, confirming the functional contribution of microglia-derived Hex to neuronal ganglioside degradation (Figure 8m, Extended Data Figure 17k).

[0256] To explore uptake mechanisms, Hexb'7' NPCs were co-treated with inhibitors: 5-(N-ethyl-N- isopropyl) amiloride (EIPA) and Wortmannin (micropinocytosis), or mannose-6-phosphate (M6P receptor blockade). All significantly reduced Hex uptake (Figure 8n), suggesting two parallel routes: macropinocytosis and M6PR-mediated endocytosis. These findings were further validated in Hexb'7' fibroblasts (Extended Data Figures 17l-o).

[0257] To test this mechanism in tissue, microglia were depleted in organotypic hippocampal Hexb'7' or Hexb+7' slices using clodronate, then reintroduced Hexb-competent microglia (Extended Data Figure 17p,j). Upon depletion, the Hex activity completely dropped, and strongly correlated with IBA1 + microglia numbers at d17, clearly pointing towards microglia as the main source of secreted Hex within the murine CNS (Extended Data Figure 17q-s, u). At d17, HEXB was observed in neurons of chimeric Hexb'7' slices, confirming microglial supply (Extended Data Figure 17t). Together, microglia constitutively secrete Hex through the classical secretory Golgi-dependent pathway. The enzyme is endocytosed by neurons through macropinocytosis and M6P receptor-mediated endocytosis, trafficked to lysosomes and facilitates GM2 degradation.

[0258] Hexb in MLCs prevents neurodepeneration

[0259] Having shown that microglia can restore neuronal Hex activity ex vivo, next it was tested whether microglial replacement could provide therapeutic benefit in vivo. To this end, Hexb'7' mice underwent microglia depletion by using the CSF1 R inhibitor BLZ945, followed by transplantation with Cx3cr1GFP7+:Hexb+7' bone marrow (Extended Data Figures 18a-d). Recipient mice showed high degree of myeloid cell engraftment after P245 (Figures 9a, b, Extended Data Figure 18e). Strikingly, chimeric Hexb'7' mice displayed normalized survival, mitigated motor symptoms and stable body weight (Figures 9c-e, Extended Data Figure 18f). Brain homogenates exhibited restored Hex activity and markedly reduced of GM2 accumulation (Figures 9g,h,i, Extended Data Figures 18g). Neurons in transplanted animals also regained Hex activity and HEXB immunoreactivity, confirming microglia as a sufficient extrinsic source. (Figures 9j,k). Importantly, without BLZ945-mediated niche depletion, transplantation alone had only negligible effects on disease outcome, indicating the necessity of an empty microglia niche (Figures 9b-e). Indeed, behavioral improvement significantly correlated with the proportion of engrafted Hexb-competent MLCs (Figure 9f). Of note, donor-derived MLCs exhibited approximately 60% (61 .49 ±2.39%) of Hexb mRNA expression and 70% (70.72 ± 0.98%) of Hex enzymatic activity compared to residual endogenous microglia (Figure 9I, Extended Data Figure 18h). Moreover, initiating treatment neonatally further enhanced outcomes (Extended Data Figures 18i-l), collectively underscoring the therapeutic potential of early intervention and genetically enhanced donor cells.

[0260] To analyze transcriptional changes, snRNA-seq of thalamic nuclei from transplanted and untransplanted Hexb'7' and Hexb+7' mice were performed (Extended Data Figures 18m-o). Unsupervised clustering identified three major microglia clusters: c1 was enriched for homeostatic genes (Tmeml 19, P2ry12, and SalH). c2 showed elevated expression of disease-associated genes (Spp1 , Gpnmb, Ctsb, and Cst7), and cO consisted primarily of donor-derived MLCs with a distinct profile (Extended Data Figures 18p-t). Host microglia in transplanted Hexb'7' brains showed reduced c2 occupancy and increased expression of homeostatic genes (Extended Data Figures 18u , v), suggesting partial transcriptomic normalization through MLC engraftment. In sum, these data strongly suggest that donor-derived MLCs restore lysosomal function, limit GM2 buildup, and help re-establish homeostatic microglial states, offering a clinically applicable strategy for Sandhoff disease treatment.

[0261] Shared disease pattern in Sandhoff brains

[0262] To compare the murine findings to the human disease, histopathological analysis of CNS tissue of Sandhoff disease patients and aged- and sex-matched controls were performed. IBA1 immunohistochemistry revealed large foamy microglia with retracted processes in Sandhoff disease specimens only (Figure 10a), particularly enriched in the cerebellum (Extended Data Figure 19a). These microglia showed elevated levels of KIM1 P, p22phox, lysozyme, and LAMP2, consistent with lysosomal activation as observed in mice (Extended Data Figure 19b). Hematoxylin and eosin (H&E) stainings revealed excessive intracellular inclusions and hypercellular Virchow-Robin spaces (Extended Data Figures 19c,d). Inclusions were Luxol-Fast-Blue (LFB)+and mostly found in neurons and microglia (Figure 10a, b). As in mice, microgliosis in Sandhoff individuals coincided with axonal swelling and damage in the thalamic nuclei (Figure 10a) with diminished axonal density in the thalamic white matter (Extended Data Figure 19e). SMI31 immunohistochemistry revealed phosphorylated neurofilament accumulation in neuronal somata, while SMI35 and SMI312 revealed axonal swellings and impaired axonal transport (Extended Data Figure 19f).

[0263] Then microglia were characterized transcriptionally by snRNAseq on 14,657 single nuclei from thalamic specimens. Cell type annotation identified different structural CNS cell types including astrocytes, oligodendrocytes, immune cells, OPCs, and neurons, and others (Figure 10b, Extended Data Figure 19g). Subsetting and reclustering revealed four myeloid cell clusters (Figure 10c). c0-2 as microglia (CX3CR1 , P2RY12, NAV3) and c3 as CAMs (F13A1 , MRC1 , CD163, LYVE1) (Figure 10d). Homeostatic microglia were concentrated in cO, whereas disease-linked cells appeared in c1 and c2 (Figure 10c, Extended Data Figure 19h). They strongly downregulated CX3CR1 , P2RY12, TMEM119 and SALL1 and upregulated GPNMB, MS4A7, MYO1 E, SPP1 and LPL - mirroring the murine response (Figures 10d-f, Extended Data Figure 19i). GO analysis inflammatory signaling (c1) and lysosomal / autophagy pathways (c2) (Figure 10d). Ultimately, untargeted lipidomics and spatial MALDI-MSI of patient brain tissue revealed GM2 species — such as GM2 34:1 ;O2 to 40:1 ;O2 — in cortex and thalamus, closely matching the murine GM2 profile (Figure 10g, Extended Data Figures 19j,k). In conclusion Sandhoff patient brains recapitulate the transcriptional, histological, and lipidomic signatures seen in Hexb-deficient mice, confirming a shared disease mechanism.

[0264] DISCUSSION

[0265] The study describes a novel microglia-neuron connection that ensures normal brain homeostasis (Extended Data Figure 20). By combining unbiased lipidomics, single-cell transcriptomics, spatial lipid imaging, and novel genetic models, a functional relationship in which the microglial lysosomal enzyme Hex regulates neuronal lipid balance is defined. Themicroglial core gene expression was initially monitored across models of neurodegeneration and demyelination. While expression of Tmem119, Siglech and SalH was reduced, Cx3cr1 and Hexb remained stable. Using HexbtdTreporter mice, the microglial specificity and developmental regulation of Hexb was confirmed. Although neurons expressed trace Hexb mRNA (~200-fold lower), its functional significance was unclear.

[0266] Global deletion of Hexb induced early-onset neurodegeneration, consistent with human Sandhoff disease. It has been described that a dysfunctional Hex leads to ganglioside storage within neurons and subsequent neuronal cell death with reactive microglia perpetuating a neurotoxic milieu. The histological and single-cell data show that microglial activation precedes astrocytic or neuronal changes, suggesting microglia are early responders. Along with the striking finding that full microglia replacement halts disease progression, this supports classifying Hexb-deficiency as a novel microgliopathy.

[0267] The transcriptional profile of Hexb'7' microglia partially resembled activated states seen in other neurodegeneration models, with upregulation of Apoe, Ctsb, Csf1 , Igf1 , Lyst and Gpnmb, alongside distinct markers such as Apobecl , Flt1 , Colec12, Adam33 orAtp6v0d2. Gpnmb, the most upregulated gene, encodes a transmembrane glycoprotein induced in lipid-laden macrophages and linked to anti-inflammatory and tissue repair roles. Importantly, phagocytosis and autophagy pathways, both commonly disrupted in various lysosomal storage disorders (LSDs), were strongly altered in Hexb'7' microglia, underscoring the enzyme’s key role in lysosomal function.

[0268] Another strongly upregulated gene in Hexb-deficient microglia was Ms4a7, previously considered a marker of peripheral myeloid origin. However, the new data suggest that Ms4a7 reflects activation state rather than ontogeny, highlighting the limitations of using single-gene markers in distinguishing resident from peripherally derived myeloid populations in the CNS.

[0269] To understand how microglial enzyme loss causes neurodegeneration, the mechanism of Hex transfer was dissected. Microglia secrete the enzyme via the Golgi pathway into the extracellular space, where it is taken up by neurons to degrade GM2 gangliosides. Lipidomics confirmed GM2 as the most excessively accumulated lipid. Notably, GM2 species with longer ceramide backbones increased with disease progression, reflecting postnatal developmental shifts in ganglioside composition.

[0270] GM2 accumulation in both mouse and human Hexb-deficient brains correlated with strong microgliosis, suggesting a causal link between these two events. Indeed, GM2 — but not GM1 or GM3 — induced proinflammatory cytokines in microglia, indicating a specific immune response. Interestingly, while GM1 gangliosidosis is also associated with pronounced microglia activation and cytokine release, this effect does not appear to stem from direct GM1 recognition. In fact, GM1 has been shown to exert anti-inflammatory effects on microglia. MGL2 was identified as the key receptor mediating GM2’s effects. Expressed in dendritic cells, macrophages, and microglia, MGL2 binds terminal GalNAc residues. In general, its signaling is context- and ligand-dependent with evidence for both pro- and anti-inflammatory responses.

[0271] To pinpoint disease-driving cell types in vivo, conditional knockouts targeting Hexb in specific compartments were generated. Contrary to the expectations, deleting Hexb in microglia alone did not induce disease. Only combined depletion in neurons and microglia recapitulated the full phenotype, indicating functional redundancy. Similar compensation has been observed in other contexts — for example, Grn deletion in microglia alone does not trigger CNS pathology. Overall, while the absence of a functional Hexb gene in microglia alone does not cause neurodegeneration, the presence of a functional Hexb gene only in microglia is sufficient to prevent neurodegeneration. However, microglia likely contribute to pathology by amplifying inflammation. IL-6, TNF, CCL3, CCL4, and CCL17 release from microglia upon GM2 exposure is described and upregulated phagocytic pathways in microglia potentially promoting disease progression is identified. A similar mechanism has been described in Gaucher disease, where lipid-accumulating microglia phagocytose live neurons, and, more broadly, lipid metabolic dysfunction has been tied to neuroinflammation.

[0272] Beyond GM2, several lipids accumulating in the brains of Hexb mutant mice, including GA2, GalNAc-GM1 36:1 -02 (also known as asialo-GM2), and bis(monoacylglycero) phosphate (BMP) 44:12 were identified. The spatiotemporal lipid map revealed pathological hotspots, but the direct link between ganglioside buildup and neuron loss remains unclear. A recent study implicated neuron- intrinsic cGAS-STING signaling in Hexb-related neurodegeneration, but the specific vulnerability of neuronal subtypes to GM2 stress warrants further investigation.

[0273] Until currently, no curative therapies exist for Sandhoff disease. Besides symptomatic treatment, currently available and approved LSD therapies — enzyme replacement therapy (ERT), substrate reduction therapy (SRT), or chaperone therapy — are often prohibitively expensive and primarily serve to slow disease progression rather than halt it. Notably, ERT fails to effectively address the CNS involvement seen in many LSDs, as the BBB prevents administered enzymes from reaching the brain parenchyma. Gene therapy, however, holds promise: AAV-based delivery of Hexb cDNA has shown success in mice, and these findings have been translated to human GM2 gangliosidoses, but achieving full CNS coverage remains challenging. Bone marrow transplantation (BMT) offers an alternative but has shown limited benefit in GM2 gangliosidosis, likely due to insufficient engraftment of enzyme-competent myeloid cells. Previous studies have shown before that CNS preconditioning (e.g., irradiation) is required for myeloid cell engraftment, which can be significantly augmented in several inflammatory and neurodegenerative models. Still, microglia replacement rarely exceeds 30 %. However, higher microglia and CAM turnover with circulating blood cells have been described recently in aged patients without CNS diseases. In a mouse model of Sandhoff disease, Chen et al. improved the engraftment of Hexb+MLCs by first depleting resident microglia pharmacologically, then transplanting cultured murine microglia into the open niche, which prevented disease symptoms and attenuated neurodegeneration. Building on these findings, clinically applicable BMT combined with microglia depletion were used to efficiently replace microglia and demonstrate that donor cells restored enzymatic activity, reduced GM2, and shifted microglial transcription toward homeostasis. Early intervention further improved outcomes. This novel treatment approach might be a new potential therapeutic option for microglia-mediated human gangliosidoses as well. Similarly, the detailed dissection of the microglia-neuron crosstalk which regulates GM2 turnover could inform strategies for cell replacement therapy in Sandhoff disease and other gangliosidoses. In summary, the findings expand the understanding of microglial functions in the healthy CNS to include the homeostatic regulation of neuronal membrane components.

[0274] MATERIAL AND MEHTODS

[0275] Mice: Hexb'' mice (B6.129S-Hexbtm1 Rlp / J) were purchased from Jackson Laboratories (#002914) and backcrossed for five generations with C57BL / 6J mice. Hexbmmice B6.Hexbtm1c(EUCOMM>Hm9U / H) were generated by crossing the B6.Hexb,m1a<EUC0MM)Hmgu / H line from the MRC Harwell Institute with the Flp-FRT line (129S4 / SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym / J). Hexbmimice were further crossed with Cx3cr1Cre(B6J.B6N(Cg)-Cx3cr1fml7^J,m9 / J)Or A / esCre(B6.Cg-Tg(Nes-cre)1 Kln / J) mouse lines. Cx3cr1GFP(B6.129P2(Cg)-Cx3cr7fm'Yf / J) mice served as bone marrow donors. In addition, Cx3cr1GFP, Thy1GFP(B6.Tg(Thy1-EGFP)MJrs / J), and Hexbfdr(B6N.Hexbem1Mp) mice were used for imaging analysis. Littermates were used as controls in all experiments. For all experiments, mice were randomly assigned to experimental groups based on their genotype. No statistical methods were used to predetermine sample sizes. For the analysis of different neurodegenerative and demyelinating disorders, the following mice (and brain regions) were used. SOD1 mice (B6.Cg- Tg(SOD1*G93A)1Gur / J; spinal cord) and R6 / 2 mice (B6CBA-Tg(HDexon1)62Gpb / 3J; striatum) were purchased from Jackson Laboratories (#004435, #006494). In addition, 5xFAD mice (B6.Cg- Tg(APPSwFILon,PSEN1*M146L*L286V)6799Vas / Mmjax; hippocampus) and APP23 mice (B6.Cg- Tg(Thy1-APP)3Somm / J; cortex) were used. Cuprizone-induced demyelination was achieved by feeding mice for 5 weeks with 0.25% (wt / wt) cuprizone (C9012, Sigma-Aldrich) in the ground breeder chow (corpus callosum). Wild type female mice on C57BL / 6N background were used as controls.

[0276] Mice were housed under a 12-h light / 12-h dark cycle and at temperatures of 18-23 °C with 40-60% humidity, with food and water provided ad libitum. Diseased mice received wet food placed on the cage ground. All animal experiments were approved by the local administration (Regierungsprasidium Freiburg, approval numbers G-17 / 34, G-21 / 020, and G-22 / 035) and were performed in accordance with the respective national, federal, and institutional regulations. Upon weight loss (> 10% loss) or occurrence of an impaired righting reflex (> 5 sec) diseased mice were euthanized and the age was recorded.

[0277] Behavioral testing: Rotarod Motor coordination was assessed using the rotarod assay. The assay was conducted using a Rota-Rod (Model 47650, Ugo Basile) with accelerating speed (accelerated from 3 to 40 r.p.m. over 300 s). The mice were trained on the accelerating rotarod one day before the first recorded testing. The latency to fall off the rotarod was recorded three times a week until the end of the observation period. Only one trial was conducted per day. A full passive rotation or falling of the rotarod was considered a failure and recorded as “latency to fall”. Grip strength test: Muscular strength was assessed with the grip strength test using a Grip-Strength -Meter (Mains). Mice were grasped at their tail and placed on a slight oblique grid with all four limbs. Now, the tail was gently and continuously pulled backward. The maximum force was automatically recorded when the mouse lost its grip. Each mouse was tested three times; the best trial was recorded.

[0278] Human specimen and ethics: Human tissues were obtained from the NIH Neurobiobank at the University of Maryland, Baltimore, MD. Samples were shipped on dry ice and stored at -80°C until used. The examination of adult autopsy tissues was carried out with supervision from the Research Ethics Committee at the University Freiburg Medical Center, following protocol numbers 10008 / 09 and 472 / 15, as well as oversight from local committees affiliated with the National Institutes of Health (NIH) bio banks. Written informed consent was obtained from the patients or their legal guardians prior to the procedures.

[0279] Nuclei isolation from frozen tissues: Nuclei isolation was performed as previously described. In brief, a small tissue fragment was homogenized and incubated in 500 pL of ice-cold nuclei EZ lysis buffer (NUC101-1 KT, Sigma-Aldrich) for 5 minutes. After filtration through a 70 pm filter (B60160056, Miltenyi) and centrifugation at 500g for 6 minutes at 4°C, the supernatant was removed. Subsequently, 1 mL of ice-cold EZ lysis buffer was added, followed by incubation on ice for 5 minutes. After centrifugation, the supernatant was discarded, and the pellet was incubated for 5 minutes with 0.5 mL of nuclei buffer (1X DPBS (D8537, Sigma-Aldrich), 1 % bovine serum albumin (BSA) (130-091-376, Miltenyi), 0.2 U / pl RNase inhibitor (M0314L, New England Biolabs)). After gentle pipetting and centrifugation, the washing step was repeated with 1 mL of nuclei buffer. Following another centrifugation step, the supernatant was removed, and the nuclei were incubated for 10 minutes with a staining mix containing 4,6-diamidino-2-phenylindole (DAPI) (10 pg / mL), and for human samples Anti-Olig2 Alexa-488 (1 :100) (ab225099, abeam), and anti-NeuN Alexa-647 (1 :100) (ab190565, abeam) antibodies in a total volume of 200 pL of nuclei buffer. After additional centrifugation, the supernatant was discarded, and the pellet was resuspended in 300 pL of nuclei buffer, filtered through a 40 m cell strainer (14-100-150, ThermoFisher), and subjected to FACS nuclei sorting. Nuclei were sorted on a MoFlo Astrios (Beckman Coulter) or BD FACSAria III machines (BD Bioscience).

[0280] 10x Genomics droplet-based single-nucleus library preparation: Per reaction, up to 40.000 DAPI+murine nuclei or DAPI+Olig2'NeuN_human nuclei were sorted into Eppendorf tubes. Single nuclei were packaged into droplets and lysed, followed by barcoding through mRNA reverse transcription using the Chromium controller with the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics). cDNA amplification and library preparation were conducted following the manufacturer’s instructions. Libraries were sequenced on a NextSeq1000 (Illumina) appropriate to reach 20,000 reads per cell. The resulting fastq files were further processed using the Cell Ranger v7.1.0 pipeline (1 Ox Genomics) for demultiplexing, read alignment either to the mouse (GRCm38, mm10) or human genome (GRCh38p13, Gencode v35, hg38), and gene count determination.

[0281] Doublet detection, quality control, and analysis of the single-nucleus transcriptomic data: Mouse and human transcriptomic data was analysed in RStudio (Build 421), with R programming language version 4.3.2. Filtered counts matrices were loaded with Seurat v.5.0.3. Doublets were excluded using the scDblFinder package v.1.16.0. Therefore, the Seurat object was transformed into a SingleCellExperiment object using the as. SingleCellExperiment function, the scDblFinder function was run, and the original Seurat object was filtered for cells classified as ‘singlet’. The data from different experiments was merged into one Seurat object (merge). For mouse samples, a three-step procedure for strict quality control was applied. First, only nuclei with < 0.1 % of mitochondrial transcripts, <0.1% haemoglobin transcripts, and between 300 and 4500 genes expressed were retained. Second, after an initial normalization and integration (see below), cluster expressing “debris marker” defined by the FindMarkers function were excluded. Debris marker were previously identified by loading raw counts matrices and, after normalization and integration, identifying marker genes (FindMarkers) of cluster 0 containing empty partitions and low quality cells (Tubala, Hspa8, Atp6v0c, Tubb2a, L / bb). Third, nuclei were manually inspected using the Feature Scatter a nd CellSelector functions to visualize known cell-type-specific marker genes. Nuclei expressing more than one cell-type-specific marker were filtered. Since the mouse dataset contained nuclei of all CNS cell types, the amount of expressed genes differ between cell-types and clusters. Thus, an additional round of nuclei exclusion was performed based on expressed genes (nFeature_RNA) for annotated cell types as follows: astrocytes (500 - 2,000 expressed genes), immune cells (300 - 1 ,800 expressed genes), VLMCs (800 - 2,000 expressed genes), ependymal cells (300 - 2,000 expressed genes), oligodendrocytes (1 ,800 - 3,800 expressed genes), OPCs (600 - 2,800 expressed genes), COPs (600 - 2,800 expressed genes), neurons (1 ,300 - 3,800 expressed genes). For human samples, nuclei with at least 300 and fewer than 4,500 detected genes and below 2% mitochondrial transcripts were included. In addition, myeloid cells with more than 2500 detected genes were discarded. Visualization was achieved using the DimPlot function. After quality control, the data was normalized (NormalizeData) and scaled on the 2,000 most variable features (FindVariableFeatures, ScalaData). Linear dimensional reduction was performed using the RunPCA function. Next, the different experiments within the Seurat object were integrated using the Harmony R package v.1 .2.0 (RunHarmony). Lastly, UMAP embedding and shared nearest-neighbors graph construction were performed on the top 10 principal components (top 7 principal components for human data) (RunUMAP, FindNeighbors), and cell clusters were identified with a resolution set to 1 .2 (0.5 for human data) (FindClusters).

[0282] Cell annotations, differential gene expression analysis, and GO enrichment analysis: For every cluster, differentially expressed genes (DEGs) were calculated using the FindAIIMarkers function with logfc.threshold and min. pct arguments set to 0.25. DEGs were used for cluster annotation based on published cell-type-specific marker genes. For further analysis of the mouse data, immune cells and subsequently microglia were abstracted to generate a new Seurat object using the subset function. Re-normalization, -scaling, -integration, and -clustering was performed as described above. Again, cluster marker were calculated using the FindAIIMarkers function with logfc.threshold and min. pct arguments set to 0.25. For further analysis of the human data, myeloid cells were isolated into a new Seurat object (subset). Re-normalization, -scaling, -integration, and - clustering was performed with slight modifications: data was scaled on the 5,000 most variable features and the variables “percent.mt” and “percent.rp” were regressed out. The different patients were integrated using Harmony and UMAP embedding and shared nearest-neighbors graph construction were performed on the top 10 principal components. Then, cluster marker were calculated using the FindAIIMarkers function with default settings utilizing the MAST algorithm. The significant (adjusted p value < 0.05) cluster marker genes were subjected to Gene Ontology enrichment analysis performed with the clusterProfiler v4.10.0 package. Maker genes were transformed into entrezIDs and the enrichGO function was run on them to identify enriched biological processes. Microglia clusters were re-ordered from cluster size to altered biological function. For direct comparison of clusters FindMakers function was run with default settings utilizing the MAST algorithm. Data was visualized using DimPlot, DotPlot, FeaturePlot, and DoHeatmap Seurat functions. In addition, EnhancedVolcano package (v1.20.0) was used to generate volcano plots.

[0283] Cross-species analysis: For the comparison of mouse and human disease microglia, DEGs for mouse and human disease microglia were calculated independently by comparing disease- associated cluster with homeostatic cluster (FindMakers). DEGs were filtered for statistical significance (adjusted p value < 0.05). Mouse gene names were converted to human orthologous gene names using the R package BiomaRt (v2.58.2). Only genes that have a corresponding orthologous name were retained. Using ggplot2 package (v3.5.0), average log2(FC) values of human and mouse genes were plotted.

[0284] Bulk RNA-seq: Microglia were FACS-sorted from whole into a collection tube and then total RNA was extracted using PicoPure RNA Isolation Kit (KIT0204, Life Technologies) according to manufacturer’s protocol. The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from approximately 1 ng total-RNA. Double-stranded cDNA was amplified by LD PCR (10 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). Thereby 150 pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. The libraries were quantified using the KAPA Library Quantification Kit - lllumina / ABI Prism User Guide (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA). Equimolar amounts of each library were sequenced on an Illumina NextSeq 2000 instrument controlled by the NextSeq 2000 Control Software (NCS) v1.4.1.39716, using two 50 cycles P3 Flow Cells with the dual index, single-read (SR) run parameters. Image analysis and base calling were done by the Real Time Analysis Software (RTA) v3.9.25. The resulting .cbcl files were converted into .fastq files with the bcl2fastq v2.20 software. Fastq files were quality controlled using FastQC v0.73 and trimmed with Trim Galore! v0.6.7. Reads were mapped to the GRCm39 mouse genome using the RNA STAR aligner v2.7.8. Read counts were obtained using featurecounts v2.0.1 . Differential gene expression analysis was performed using the limma-trend pipeline v3.50. Gene Ontology (GO) enrichment analysis of DEGs was made using goseq v1 .44.0. The mentioned processes were run on the galaxy platform. PCA analysis was conducted with the ggfortify R package (v0.4.17). Heatmaps were generated using the R package pheatmap (v1.0.12). Venn diagram was generated by using previously published tools (https: / / bioinformatics.psb.uQent.be / webtoolsA / enn / ). Volcano plots were calculated with the R package EnhancedVolcano.

[0285] Flow cytometry: For blood cell analysis, one drop of blood was collected from the facial vein into FACS buffer (PBS containing 2% BSA (8076.3, Roth) and 10 mM EDTA (15575020, Invitrogen)) to prevent clotting. Blood cells were centrifuged at 300g for 5 min at 4°C. Blood cell pellet was resuspended in RBC lysis buffer (00-4333-57, Thermo Fisher) and incubated for 2 min at RT. Ice cold FACS buffer was added and cells were centrifuged again before staining. For microglia analysis, mice were anesthetized and transcardially perfused with ice cold PBS. Brains were roughly minced and homogenized with a potter tissue grinder in HBSS (14170-138, gibco) containing 15 mM HEPES (15630080, gibco) buffer and 0.54% glucose (G8769, Sigma). Whole brain homogenate was separated by 37% Percoll (P1644, Sigma) gradient centrifugation at 800g for 30 min at 4 °C (no brake). The pellet containing CNS macrophages at the bottom of the tube was then collected and washed once with FACS buffer before staining. Fc receptors were blocked with Fc Block (2.4G2, BD Biosciences) for 15 min at 4°C before incubation with the primary antibodies. Cells were stained with antibodies directed against CD11 b (1 :300, M1 / 70, BioLegend), CD45 (1 :200, 30-F11 , Invitrogen), Ly6C (1 :300, AL-21 , BD Biosciences), Ly6G (1 :300, 1A8, BD Biosciences), CD115 (1 :200, AFS98, Invitrogen), CD64 (1 :200, X54-5 / 7.1 , BioLegend), CD11c (1 :300, N418, Invitrogen), CD3e (1 :300, eBio500A2, Invitrogen), CD19 (1 :300, eBio1 D3, Invitrogen), B220 (1 :300, RA3-6B2, BioLegend), and CD206 (1 :200, C068C2, BioLegend) for 45 min at 4 °C. After washing, cells were sorted using a MoFlo Astrios (Beckman Coulter) or analyzed using a BD LSRFortessa (Becton Dickinson). Viable cells were gated by staining with DAPI. Data were acquired with FACSDiva or Summit software (Becton Dickinson). Postacquisition analysis was performed using FlowJo software, v.10.5.3.

[0286] Chromogenic immunohistochemistry and cell quantifications: Mice were anesthetized and transcardially perfused with ice cold PBS. Brains were fixed in 4 % formalin and embedded in paraffin. 3 pm paraffin sections were initially deparaffinized at 80°C for 1 h, then deparaffinized in Xylene and incubated in EnVision FLEX Target Retrieval Solution pH 6 cooking buffer (S1699, DAKO) for 40 minutes at 95°C. Endogenous tissue peroxidase was blocked in 3% hydrogen peroxidase for 10 minutes. Samples were blocked with PBS containing 5 % BSA (8076.3, Roth) and permeabilized with 1% Triton-X 100 (T8787, Sigma) for one hour. Primary antibodies were added over night at a dilution of 1 :1 ,000 for IBA1 (ab178846, Abeam), 1 :200 for Mac-3 (553322, BD Pharmingen), 1 :10,000 for GFAP (Z0344, DAKO), 1 :3,000 for APP (MAB348, Merck), 1 :500 for P2RY12 (AS-55043A, Anaspec), 1 :500 for TMEM119 (400 002, Synaptic Systems), 1 :1 ,000 for NeuN (ab104224 or ab177487, abeam), 1 :500 for HEXB (LS-B16803, LSBio) at 4°C. After three washes with PBS, biotinylated secondary antibodies (Southern Biotech) were added as follows: goat-anti-mouse 1 :200 (1031-08), goat-anti-rabbit 1 :300 (4050-08), and goat-anti-rat 1 :200 (3050- 08) for 45 min at RT. Three more washing steps were performed before incubating the sections with streptavidin peroxidase (PK-6100, Vector Laboratories) for 45 min at RT. After three more washes with PBS, slides were incubated with DAB solution: 1 drop EnVision Flex DAB Chromogen (DM827, DAKO) per 1 mL EnVision Flex Substrate Buffer (DM823, DAKO). For double-immunolabeling, the process was repeated using streptavidin-AP and Permanent Red as chromogen. Finally, the slides were counterstained with Gill’s Hematoxylin solution (11769, Morphisto). Coverslips were mounted with xylene-based Vitro-Clud mounting medium (04-0001 , Langenbrinck) or Kaisers Glycerin- Gelatine (6474.1 , Roth), respectively. For quantification, Images were taken using BZ-X810 microscope with BZ-X8000 Analyzer software (Keyence, Osaka, Japan). To assess density of cells IBA1+, Mac-3+, GFAP+cells or APP+deposits were quantified manually as previously described81. To assess HEXB+neurons, only large cells with a visible cytoplasm and prominent nucleolus were analyzed. At least three sections of a minimum of three mice were used for each analysis. Representative images were acquired with a Leica DFC450 Digital Microscope Camera. Postacquisition editing was done with Adobe Photoshop CS4 (Adobe).

[0287] Brain tissue obtained at autopsy was fixed in buffered formalin and embedded in paraffin. 3 pm thick paraffin sections were treated as previously described. Briefly, after deparaffinization in xylene, sections were transferred to 99.5% ethanol and rehydrated to distilled water using decreasing ethanol series. Staining for macrophages / activated microglia (clone KiM1 P, 1 :50), IBA1 (1 :1 ,000, ab178846, Abeam), APP (1 :2,000, MAB348, Sigma), p22phox (1 :100, sc-20781 , Santa Cruz), and phosphorylated neurofilament (SMI31 ; 1 :5000, Stemberger Monoclonals Inc.) required antigen retrieval with citrate buffer (antigen retrieval solution, Dako), whereas for lysozyme (1 :200, 18-0038, Zymed) protease pretreatment and for LAMP2 (1 :500, SA46-01 .Thermofisher) TE-buffer was used, each for 50’ in a steaming device (Braun, Germany). Neurofilament (SMI35, hypophysphorylated, and SMI312, highly phosphorylated) (both 1 :1000, Stemberger Monoclonals Inc.) required no antigen retrieval. Sections were blocked in 10% FCS for 10 min and 3% H2O2 prior to incubation with primary antibodies for 90 min at room temperature. After washing in PBS, sections were incubated with biotinylated secondary antibodies, then incubated with avidin-coupled horseradish peroxidase, and finally developed in diaminobenzidine (DAB). For KiM1 P immunohistochemistry, DAB Envision Kit (Dako) was used. Nuclei were visualized with hematoxylin. Light microscopy sections were photographed using an Olympus BX51 microscope and cellSense software (Olympus).

[0288] Fluorescent immunohistochemistry, cell quantifications, analysis, and IMARIS-based 3D reconstruction: After transcardial perfusion with ice cold PBS, brains were fixed for 5 h in 4 % formalin, dehydrated in 30 % sucrose and embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek Germany GmbH). 14 pm (for quantification), 30 pm (for representative images), or 50 pm cryosections (for 3D reconstruction) were obtained and blocked with PBS containing 5 % BSA and permeabilized with 0.5% Triton-X 100 in blocking solution (no Triton-X 100 was used for GM2 stainings). Primary antibodies were added over night at a dilution of 1 :1000 for IBA1 (ab178846, Abeam; 234 308, Synaptic Systems), 1 :500 for CD206 (MCA2235, Bio-Rad), 1 :200 for collagen IV (AB769, Millipore), 1 :500 for SOX9 (AF3075, R&D), 1 :500 for NeuN (ab104224, abeam), 1 :200 for OLIG2 (ab109186, abeam), 1 :100 for GM2 (A2575, TCI), 1 :500 for TMEM119 (400 002, Synaptic Systems), 1 :500 for P2RY12 (AS-55043A, Anaspec), and 1 :100 for CD68 (MCA1957, Bio-Rad), and -M -

[0289] 1 :500 for LAMP1 (PA1-654A, Invitrogen) at 4°C. Secondary antibodies were purchased from Thermo Fisher Scientific and added as follows: Alexa Flour® 405 1 :500, Alexa Flour® 488 1 :500, Alexa Flour® 568 1 :500 and Alexa Fluor® 647 1 :500 for 90 min at 4°C. For nuclear counterstaining, DAPI was added for 30 min at RT. Coverslips were mounted with Mowiol (0713.2, Roth). For quantification, images were taken using the conventional fluorescence microscope BZ-X810 (Keyence, Osaka, Japan). To assess density of cells, numbers of IBA1+CD206- (microglia) or CD206+cells (pvM<t> and mM<t>) were quantified. Microglia and pvM<t> were normalized to the area of the region of interest and expressed as cells mm-2. mM<t> were normalized to the length of the leptomeninges indicated by collagen IV or laminin immunofluorescence and finally expressed as cells / mm. To assess labeling for tdTomato, IBA1+P2RY12+microglia, CD206+pvM<t> and SOX9+astrocytes, NeuN+neurons, OLIG2+oligodendrocytes were counted and analyzed. At least three sections of a minimum of three mice were used for each analysis. Representative confocal images are taken with the TCS SP8 X (Leica) using a 20x or 63x objective, respectively. Postacquisition editing was done with LAS X software (Leica) and Adobe Photoshop CS4 (Adobe). For quantification of the lysosomal ganglioside burden, lysosomal compartments were segmented based on LAMP1 fluorescence. Within these LAMP1+regions, the mean fluorescence intensity (MFI) of the GM2 signal was measured. For each cell (defined by IBA1 or NeuN signal), the GM2 signal across all lysosomal regions was summed to obtain the total lysosomal GM2 fluorescence per cell. This total lysosomal GM2 signal was then normalized to the corresponding cell area to allow comparison between cell types. Image analysis was performed using Imaged (v1.54g). For 3D reconstruction of microglia and lysosomes, sections were co-stained with IBA1 and CD68. IBA1+parenchymal cells were selected and imaged with the TCS SP8 X (Leica) using a 63x objective with Z-stacks of 0.3 pm. 3D reconstructed cell images and statistical read out was obtained using Imaris software v9.6.0 (Bitplane).

[0290] Lipid measurement by LC-MS: For the extraction of lipid for liquid chromatography mass spectrometry (LC-MS), frozen mouse brains were thawed on ice, for 1 min, and 10 mg of cortical brain tissues were mechanically homogenized in in 1 mL 20% methanol (Carl Roth, 8399.1). Cultured NPCs were washed three times with PBS, collected and mechanically homogenized in 0.1 mL 20% methanol. Next, 500 pL of the homogenate was diluted in 750 pL ddb^O plus 2.5 mL 1- butanol, vortexed for 1 min, and centrifuged for 1 min at 20 000 g to separate phases. The top layer (butanol) was transferred to a 4 mL glass tube. 1 mL water-saturated 1 -butanol was added to the remaining aqueous phase, vortexed for 1 min, and centrifuged for 1 min at 20 000 g to separate phases. The top layer was combined with the butanol phase obtained in the first extraction. The butanol phase was dried in a speedvac. Just prior to measurement, the pellets were resuspended in 50 pL of a 2:1 :1 mixture of 2-propanol, acetonitrile, and ddb^O. Non-targeted measurement of lipids by LC-MS was carried out as described previously using an Agilent 1290 Infinity II UHPLC in line with a Bruker Impact II QTOF-MS operating in negative ion mode. Briefly, scan range was from 50 to 1600 Da. Mass calibration was performed at the beginning of each run. LC separation was on a Zorbax Eclipse plus C18 column (100 x 2 mm, 1 .8 pm particles) using a solvent gradient of 70% buffer A (10 mM ammonium formiate in 60:40 acetonitrile:water) to 97% buffer B (10 mM ammonium formiate in 90:10 2-propanol:acetonitrile). Flow rate was 400 pL / min, autosampler temperature was 5 °C and injection volume was 2 pL. Data processing including feature detection, feature deconvolution and annotation of lipids was performed using MetaboScape (version 2023b). MALDI Mass Spectrometry Imaging (MSI): Fresh-frozen mouse and human brain tissue were sectioned at 10 and 20 pm thickness, respectively, with a Leica CM1950 cryostat (Leica Biosystems) at -18°C chamber- and specimen head temperature. Sections were thaw-mounted onto ITO slides (Bruker Daltonics) and stored at -80°C. For further use, slides were brought to room temperature and dried for 15 minutes in a vacuum desiccator. Optical images were acquired using a Tissue Scout slide scanner (Bruker Daltonics). For matrix spray-coating, 2,5-Dihydroxyacteophenone (DHAP) was suspended in 7:3 (v / v) acetonitrile (ACN):H2O at 10 mg / mL. The suspension was vortexed and sonicated until solid DHAP was fully dissolved. Then 0.1 % (v / v) trifluoroacetic acid (TFA) was added, and the mixture was vortexed. The matrix was deposited with an M5 TM-Sprayer (HTX Technologies). Temperatures of the spray nozzle and tray were 75°C and 35°C, respectively. The spraying parameters were as follows: Spray Nozzle Velocity: 1200 mm / min; Flow Rate: 0.1 mL / min; No. of Passes: 10; Track Spacing: 2 mm; Pattern: HH; Pressure: 10 psi; Gas Low Rate: 2 L / min; Nozzle Height: 40 mm; Drying Time: 0s. Prior to MSI data acquisition, external mass calibration was achieved using red phosphorus (RedP) clusters Pn (n = 13-61 in intervals of 4) and an enhanced-quadratic calibration model. MALDI MSI was carried out on a timsTOF fleX system (Bruker Daltonics) equipped with a smartbeam 3D 10 kHz laser, TimsControl 5.0(4.1), and fleximaging v7.4(7.2) software (Bruker Daltonics). Data was acquired in negative ion mode (m / z range of 300-2500) with 200 laser shots per pixel, 10 kHz laser frequency and lateral step size 40 pm. The Ion Transfer parameters were as follows: MALDI Plate Offset 50 V, Deflection 1 Delta -70 V, Funnel 1 RF 400 Vpp, isCID Energy -0.0V, Funnel 2 RF 400 Vpp, and Multipole RF 380 Vpp. Collision Cell parameters: Collision Energy 10 eV, and Collision RF 2000 Vpp. Quadrupole parameters: Ion Energy 5 eV, and Low Mass m / z 320. Focus Pre TOF parameters: Transfer Time 105 ps, and Pre Pulse Storage 12 ps. For the human brain tissue sections, the internal standard (IS) SM4 35:1 ;O2 (C41 H79NO11 S, [M-H]-; m / z 792.530107) was used for internal lock-mass calibration.

[0291] MSI data evaluation and visualization: MSI data (centroided) was imported into SCiLS Lab 2024a Pro (Bruker Daltonics) and root mean square-normalized. Data was then exported as imzML file and uploaded to www.metaspace2020.eu for annotation of putative metabolites using the following settings: m / z tolerance 5 ppm, Analysis Version v2.20230517 (META-SPACE ML https: / / www.biorxiv.Org / content / 10.1101 / 2023.05.29.542736v1), databases SwissLipids-2018-02-02 and LipidMaps-2017-12-12. For ganglioside annotation, an additional in-house library based on theoretical masses was utilized. Average peak intensities were exported from SCiLS Lab, z-score transformed, and visualized as heat map via R. Ion images were exported from SCiLS Lab in viridis color scale, within a mass window of ±12 ppm.

[0292] Western Blot: Cells were lysed in lysis buffer (50 mM HEPES pH 7.4, 40 mM NaCI, 2 mM EDTA, 1.5 mM NaVO4, 30mM NaF, 10 mM Sodium pyrophosphate, 10 mM sodium beta glycerophosphate, and protease inhibitors (Sigma Aldrich, #A32965)) supplemented with 1 % Triton X-100. Cell culture supernatant was diluted at 1 :1 with lysis buffer. Protein lysates were resolved by 4-12 % SDS-PAGE at 80-120 V. Resolved proteins were transferred for 90 min at 100V to methanol-pretreated PVDF membranes to be further analyzed by immunoblotting. Membranes were blocked with 5 % non-fat dry milk prepared in TBST (Tris-buffered saline with 0.1 % Tween 20) for 1 h at RT, then incubated overnight with the following primary antibodies diluted in TBST supplemented with 1% milk at 4°C on a rotor: anti-TSG101 (ab125011 , abeam, 1 :2000), anti-GAPDH (2118, Cell Signaling, 1 :1000), anti- His (66005-1-lg, Proteintech, 1 :5000). Following incubation, membranes were washed with TBST three times for 5 min each, before incubating with the appropriate secondary antibodies diluted 1 :2000 in 1 % BSA containing TBST for 1 hour at room temperature. Membranes were then washed three times with TBST before being visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (34579, Life Technologies).

[0293] Hex activity assay: For the Hex assay, an established protocol was followed. In brief, whole brains were homogenized with a potter tissue grinder in KPBS (136 mM KCI, 10 mM KH2PO4, pH 7.25). The homogenate was centrifuged for 2 min, 1000g, 4°C. The brain homogenate, FACS-sorted microglia or bead-purified neurons were lysed with KPBS containing 1 % Triton X-100 for 10 min on ice. In a total volume of 40 mL per reaction, the cell lysates were incubated at 37°C in a 10 mM sodium citrate buffer (pH 4.2) containing 2 mM 4-methylumbelliferyl-2-acetamido-2-deoxy-b-D- glucopyranoside (MUG; 69585, Sigma). The reaction was stopped by adding 5 volumes of a 0.2 M glycine / 0.2 M Na2CO3 solution. The amount of liberated 4-methylumbelliferone was determined fluorometrically at an emission wavelength of 440 nm after excitation at 365 nm.

[0294] Primary microglia cell culture: Primary microglia were cultured as previously described. In brief, P0-2 newborn mouse pups were decapitated, and the brain was removed and placed in ice cold dissection media (HBSS (24020117, Gibco), 10 mM HEPES (15630080, Gibco), 35 mM glucose, 100 U / mL Penicillin-Streptomycin (15140122, Gibco). Meninges were removed, and cortices were microdissected and placed in 30 mL fresh dissection media. 1.5 mL trypsin (15090046, Gibco) was added and incubated for 15 min at 37°C. After incubation, 1 .2 mL trypsin inhibitor (T6522, Sigma, 1 mg / mL) was added and incubated for an additional 1 min. Then, 750 pL DNase (DN25, Sigma, 10mg / mL) was added to digest sticky DNA. Samples were centrifuged at 400g for 5 min. The supernatant was discarded, and the pellet was triturated with 5 mL microglia culture media (DMEM (11995065, Gibco), 10% heat inactivated FBS (10270106, Gibco), 100 U / mL Penicillin-Streptomycin) using a 1 mL pipet tip. Homogenate was centrifuged again at 400g for 5 min, the supernatant was aspirated, and the pellet was resuspended in 5 mL culture media. Cell density was determined using a hemocytometer. Cells were plated in Poly-D-Lysin-hydrobromid (PDL)-coated (P6407, Sigma) T- 75 flasks at a density of 50.000 cells / mm2(approx. 3-4 million cells per flask). Cells were incubated in a cell culture incubator with 5% CO2, 100% humidity, and 37°C. The following day, the cell culture medium was replaced to remove dead cells and debris. Then, the cell culture medium was changed every five days. On day 10, microglia were collected through vigorously taping the flasks and collecting the floating cells in the medium. The resulting cells are >95% microglia and were used for downstream experiments.

[0295] In vitro ganglioside stimulation: GM1 (Cay19579, Biomol), GM2 (G8397, Sigma), or GM3 (860058P, Sigma) were dissolved in chloroform:methanol (2:1 ; 6340.1 , 8388.1 , Roth) and stored at - 80°C. On the day of the experiment, gangliosides were diluted with 2-propanol (20842.312, VWR) to reach the desired concentrations and added to 96-well plates. Coating was achieved through evaporating. Then, primary microglia were added at a density of 1 x 104cells / well and incubated for 16h. The supernatant was subjected to cytokines measurement. Microglia cells were fixed in 4% formalin. For MGL blocking assay, primary microglia are pretreated with either anti-MGL antibody (HM1081 , 10 pg / mL, Hycult Biotech), isotype control IgG (02-9688, 10 pg / mL, Thermo Fisher), GalNAc (A2795, 50 mM, Sigma) or EGTA (3054.1 , 10 mM, Roth) for 30min at 37 °C before the ganglioside stimulation. Cytokine and chemokine measurement: IL-1 a, IL-1 p, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1 , IFN- p, IFN-g, and TNF, as well as CCL2, CCL3, CCL4, CCL5, CCL11 , CCL17, CCL22, CXCL1 , CXCL5, CXCL9, CXCL10, and CXCL13 in the supernatants of stimulated primary microglia were quantified using the LEGENDplex Mouse Inflammation Panel (13-plex) (BioLegend, 740446) and the LEGENDplex Mouse Proinflammatory Chemokine Panel (13-plex) (BioLegend, 740451) according to the manufacturer’s instructions. Data was acquired using a BD LSRFortessa (Becton Dickinson) and analyzed with LEGENDplex™ Data Analysis Software Suite (BioLegend).

[0296] In vitro exocytosis inhibitor treatment: Primary microglia were generated as described above, plated at a density of 1 x 104cells / well into a 96-well-plate, and incubated 24h in a cell culture incubator. Then, medium was removed and replaced with Golgicide A (from 10mM stock in DMSO, HY-100540, MedChemExpress), Vacuolin-1 (from 10mM stock in DMSO, HY-118630, MedChemExpress), lonomycin (from 5mM stock in DMSO, I24333, Invitrogen), Brefeldin A (from 50mM stock in DMSO, HY-16592, MedChemExpress), Thapsigargin (from 50mM stock in DMSO, HY-13433, MedChemExpress), BAPTA-AM (from 50mM stock in DMSO, HY-100545, MedChemExpress), or EGTA (from 0.5M stock in ddH20, pH7.5, 3054.1 , Roth) diluted in microglia medium, and incubated for 4h. After incubation, supernatant was removed and Hex activity assay was performed. Baseline values from a medium-only control were subtracted from all other values and then normalized to the untreated, DMSO-only condition. Cell viability was tested FACS-based for all conditions and viability > 90% was confirmed.

[0297] Primary NPC culture and Hex uptake assay: A single-cell suspension from P2 newborn mouse pups was obtained as described above. Cells were plated at a density of 0.5 - 1 x 104cells per well in a Poly-L-Lysine (PLL; A-005-C, Merck)- and laminin (11243217001 , Merck)-coated 96-well-plate in neuron medium (Neurobasal medium (21103049, Gibco), 1x B-27 supplement (17504044, Gibco), 2mM glutamine (G7513, Sigma), 100 U / mL Penicillin-Streptomycin). Cells were incubated with 5% CO2, 100% humidity, and 37°C. The following day, the half of the cell culture medium was replaced. Then, the cells were fed every 2-3 days through a half-medium change.

[0298] For Hex uptake assays, half of the medium was removed and replaced with neuron medium containing compounds at 2x final concentrations: EIPA (final 25 pM; from 25 mM stock in DMSO), Wortmannin (final 1 pM; from 10 mM stock in DMSO), or M6P (final 10 mM; prepared in neuron medium). After 1 h of preincubation, recombinant His-tagged HEXB (HY-P75808, MedChemExpress) was added to a final concentration of 100 nM and incubated for 6 h. Following incubation, supernatant was removed, cells were washed four times with PBS, and subjected to immunoblotting or Hex activity assay as described above.

[0299] For conditioned media experiments, fresh medium from primary wild-type microglia cultures was collected and Hex activity was quantified. Where indicated, heat inactivation was performed by incubating the medium at 95°C for 5 minutes. Conditioned medium was diluted 1 :1 with fresh neuron medium, and a half-medium change was performed. Cells were incubated for the indicated durations, then washed, lysed, and subjected to Hex activity assay.

[0300] For transwell assays, NPCs were plated at a density of 0.1 x 106cells and maintained for 4 days. A half-medium change was then performed, a transwell insert with 0.4 pm pore size (#3470, Corning) was added, and 1 x 104primary microglia were seeded into the insert in neuron medium. Microglia were preincubated for 4 h with either DMSO-containing neuron medium or with 15 pM Brefeldin A (BFA; from 50 mM stock in DMSO) prior to transfer. After 24 h of co-culture, the insert was removed, neurons were washed four times with PBS, lysed, and subjected to Hex activity assay.

[0301] For lipidomic analysis of cultured NPCs, cells were plated at 0.5 x 106in a 6-well plate. After 4 days, conditioned media was added and the cells were incubated for 48h without further medium change prior to cell lysis and lipid extraction (see above).

[0302] Primary fibroblast culture and Hex uptake assay: Primary fibroblast cultures from Hexb'' mice were established as previously described88. In brief, mice were anesthetized, transcardially perfused with ice cold PBS, and ears and approx. 5cm of the tail were cut and placed in ethanol (20821.310, VWR) for 5 min. Ears and tails were cut into small pieces and each was incubated in 2mL of digestion mix (4 mL 2.5mg / mL collagenase D (11088858001 , Merck) plus 0.25 mL 20mg / mL pronase (10165921001 , Merck)) for 90 min at 37°C on a shaker at 200 rpm. After incubation ears and tails were placed in a 70pm cell strainer into a 10 cm dish filled with 10 mL media (RPMI1640 GlutaMAX™ supplement (61870036, Gibco), 10% FBS, 50 pM 2-mercaptoethanol (31350010, Gibco), 1x MEM Non-Essential Amino Acids Solution (M7145, Gibco), 100 U / mL Penicillinstreptomycin). Tissue was grinded using a 10 mL syringe plunger. Cell suspension was centrifuged and washed twice with media. Pellets from ears and tails were resuspended in 10 mL media each, 10pL amphotericin B (15290018, Gibco) was added, and cells were plated into 10 cm cell culture dishes and incubated in a cell culture incubator with 5% CO2, 100% humidity, and 37°C. On the third day, medium was replaced to remove debris. Every consecutive three days, fibroblasts were splitted: Plates were washed once with PBS and incubated for 5 min at 37°C with 2 mL trypsin-EDTA solution (25300054, Gibco). Plates were gently taped, 10 mL fresh medium was added and the cell suspension was centrifuged at 450g. Pellet was resuspended in fresh medium, counted and seeded at a density of 2 x 105cells in new 10 cm dishes. For HEXB uptake assays, fibroblasts were seeded in a 96-well-plate at a density of cells per well and incubated 24h in a cell culture incubator. Then, medium was removed and medium supplemented with recombinant His-tagged HEXB (HY-P75808, MedChemExpress) was added and incubated for 6h. Following incubation, supernatant was removed, cells were gently washed 4 times with PBS, and subjected to immunoblotting or Hex activity assay as described above. To inhibit pinocytosis, cells were preincubated for 1 h with one of the following compounds added to the culture medium: EIPA (25 pM; from 25 mM stock in DMSO), Wortmannin (1 pM; from 10 mM stock in DMSO), M6P (10 mM; prepared in neuron medium), IGF2R-blocking antibody (20 pg / mL; AF2447, R&D Systems), or isotype control IgG (20 pg / mL; AB- 108-C, R&D Systems). Following preincubation, recombinant His-tagged HEXB was added to a final concentration of 100 nM, and cells were incubated for an additional 6 h. Cell viability was tested FACS-based for all conditions and viability > 90% was confirmed.

[0303] Organotypic hippocampal slice culture: Organotypic hippocampal slice cultures (OHSC) have been prepared from newborn P2-3 mice as previously described. In brief, mice were decapitated, the brains were removed and placed into ice cold cutting solution (HBSS (24020117, Gibco), 10 mM HEPES (15630080, Gibco), 35 mM glucose, 100 U / mL Penicillin-Streptomycin (15140122, Gibco)), and the hippocampi from both hemispheres were isolated. Isolated hippocampi were cut into 350 pm thick slices using a tissue chopper (McIlwain) and were transferred to 0.4 pm culture plate inserts (PICM03050, Millipore), placed in six-well plates containing 1 mL of culture medium (0.5X minimum essential medium (MEM; 21090022, Gibco), 25% heat-inactivated horse serum (26050088, Gibco), 25% BME basal medium (21010046, Gibco), 2 mM glutamax (35050061 , Gibco), 0.65% glucose (G8769, Sigma), and 100 U / mL Penicillin-Streptomycin (15140122, Gibco) per well. Slices were incubated in a cell culture incubator with 5% CO2, 100% humidity, and 35°C. The culture medium was changed on the first day after preparation and every two consecutive days.

[0304] Microglia replacement in OHSCs: Microglia were depleted and replenished as described before. Microglia were depleted using the macrophage toxin clodronate (233183, Merck-Millipore).

[0305] Clodronate was solved in autoclaved H2O with a concentration of 1 mg / mL. Freshly prepared OHSC were incubated with 100 pg clodronate per mL OHSC culture medium for 24 hours. Subsequently, clodronate was replaced with fresh culture medium. Microglia-depleted OHSC were kept for 10 days before microglia replenishment. Medium was changed every two consecutive days. Primary microglia were isolated as described above. After collecting primary microglia, they were resuspended in OHSC culture medium to a final density of 1000 cells per pl. 2000 cells were added on top of each microglia-free hippocampal slice. Cells were allowed to engraft for 7 days before further analysis.

[0306] Immunocytochemistry: Primary cells were fixed with 4% formalin for 15 min at RT, blocked and permeabilized with 5% BSA in PBS supplemented with 0.1% Triton X-100 for 1 h at RT. Then, the cells were incubated overnight at 4°C with anti-TuJ1 (1 :500, 302 306; Synaptic Systems), and / or anti-His (1 :1.000, MA1-21315, Invitrogen), and / or anti-LAMP1 (1 :500, PA1-654A, Invitrogen), and / or anti-Vimentin (1 : 100 , 5741 , Cell Signaling) diluted with 5% BSA in PBS, followed by incubation with secondary antibodies goat anti-chicken IgY (H+L), Alexa Fluor™ 488 (1 :500; A11039, Invitrogen), and / or donkey anti-rabbit IgG (H+L), Alexa Fluor™ 488 (1 :500, A21206, Invitrogen), and / or donkey anti-rabbit IgG (H+L) Alexa Fluor™ 568 (A10042, Invitrogen) and / or donkey anti-mouse IgG (H+L), Alexa Fluor™ 647 (1 :500, A31571 , Invitrogen) diluted in 5% BSA in PBS for 2 h at 4°C. For nuclear counterstaining, DAPI was added for 30 min at RT. Finally, the cells were visualized with the TCS SP8 X (Leica) confocal microscope using a 63x objective.

[0307] Isolation of adult neurons from mouse brain cortices: In order to isolate cortical neurons from adult mice, mice were anesthetized and transcardially perfused with ice cold PBS. The brain was taken out and brain cells isolated as described previously. A small cortical piece of cortical tissue (grain of rice) was dissected and finely minced. Tissue was incubated in 10 mL of enzyme digestion solution (75 pl Papain suspension (LS003126, Worthington) diluted in enzyme stock solution (ESS, 10 mL 10x EBSS (E7510, Sigma-Aldrich), 2.4 mL 45 % Glucose (G8769, Sigma-Aldrich), 5.2 mL 1 M NaHCO3 (AAJ62495-AP, Fisher Scientific), 200 pL 0.5M EDTA (15575020, Invitrogen), and 168.2 mL ddH2O, filter-sterilized through a 0.22-pm filter)) and equilibrated to 37°C. Samples were shaken for 30-40 min in a water bath at 37°C. Enzymatic digestion was stopped with 1 mL of 10x hi ovomucoid inhibitor solution (300 mg BSA (8076.3, Roth), 300 mg ovomucoid trypsin inhibitor (LS003086, Worthington) diluted in 10 mL PBS and filter sterilized using at 0.22-pm filter) and 20 pL 0.4% DNase (LS002007, Worthington) diluted in 10 mL inhibitor stock solution (ISS, 50 mL 10x EBSS (E7510, Sigma-Aldrich), 6 mL 45% Glucose (G8769, Sigma-Aldrich), 13 mL 1 M NaHCO3 (AAJ62495-AP, Fisher Scientific) diluted in 170.4 mL ddH2O and filter-sterilized through a 0.22 pm filter). Cells were centrifuged at 500 g, 5 minutes, 4°C. Supernatant was discarded and the pellet was subjected to neuron isolation using the Adult Neuron Isolation Kit, mouse (130-126-602, Miltenyi Biotec) following the manufacturer’s instructions.

[0308] Isolation of exosomes from cell culture supernatant: In order to remove cells, cell debris, and larger vesicles, cell culture supernatant was serially centrifuged at 300g for 10 min, 2000g for 30 min, and 10,000g for 45 min. From the remaining supernatant exosomes were isolated using the Pan EV isolation kit (130-117-039, Miltenyi Biotec) following the manufacturer’s recommendations.

[0309] BLZ945 treatment: BLZ945 hydrochloride (HY-12768A, MedChemExpress) was dissolved in 20% (2-hydroxypropyl)-p-cyclodextrin (H107, Sigma-Aldrich). In adult mice, a dose of 200 mg per kg bodyweight was applied by oral gavage for seven consecutive days. Neonates received intraperitoneal injections at P7, 9, 11 , and 13.

[0310] Bone marrow transplantation and microglia replacement: To deplete endogenous microglia, mice received BLZ945 for seven consecutive days. On the day of the transplantation, the last dose was applied. In parallel, mice were treated with neomycin (1.1 g / L; N6386, Sigma) acid water (pH 2.5) to reduce the risk of infection. Recipient mice were lethally irradiated with 9 Gray (Gy) using a RS2000 X-ray irradiator (Rad Source Technologies). Cx3cr1GFPmice served as bone marrow donors. Bone marrow was isolated from the tibias and femurs by flushing with PBS. After red blood cell removal, cells were washed, counted, and resuspended in an appropriate volume of PBS (1 x 107cells per 100 pL). Within two hours after irradiation, adult mice received donor bone marrow (1 x 107cells) via tail vein injection. Neonates were intraperitoneally injected with the same amount of donor bone marrow cells. Following the injection, treatment with neomycin acid water was continued for another two weeks. Four weeks after transplantation, mice were subjected to blood withdrawal from the facial vein to control for a proper reconstitution with donor-derived peripheral blood cells.

[0311] Intracerebroventricular injections: Mice were anesthetized and 2 pL of antibody solution (100 pg / mL; anti-MGL antibody (HM1081 , Hycult Biotech) or isotype control IgG (02-9688, Thermo Fisher)) was injected into the lateral ventricle twice weekly for three weeks in total, starting at P10. Three days after the last injection, mice were anesthetized and transcardially perfused with ice cold PBS. The brain was taken out, microglia were isolated as described above, and an equal amount of brain tissue was homogenized using a tissue homogenizer in lysis buffer (50 mM HEPES pH 7.4, 40 mM NaCI, 2 mM EDTA, 1.5 mM NaVO4, 30mM NaF, 10 mM Sodium pyrophosphate, 10 mM sodium beta glycerophosphate, and protease inhibitors (Sigma Aldrich, #A32965)) supplemented with 1 % Triton X-100.

[0312] Patch clamp electrophysiology in acute brain slices: P42 mice were deeply anaesthetized with isoflurane (5%) in oxygen-enriched air (Oxymat 3, Weinmann), and decapitated into carbonated, ice cold slicing solution. A Leica VT 1200S vibratome was used to obtain 350pm thick coronal slices from motor cortex. Slices were directly transferred to carbogenated slicing solution at 33°C for 10 minutes, and then further transferred to carbogenated standard ACSF at room temperature. After 30-60 minutes recovery time, slices were used in whole-cell patch-clamp experiments. Slicing solution contained (in mM) 93 NMDG, 93 HCI, 2.5 KCI, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4 and 0.5 CaCI2 and was calibrated to a pH of 7.3-7.4 and an osmolarity of 315 mOsm. Standard ACSF contained (in mM) 125 NaCI, 3 KCI, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 1 MgCI2 and 2 CaCI2 and was calibrated to an osmolarity of 315 mOsm. For recordings, slices were held in a chamber at 33°C and perfused with ACSF (2-4 mL / min). Cells were visualized for patching using differential interference contrast microscopy (Scientifica) with a water immersion objective (Olympus LUMPIanFLN40xW) and a CCD camera (Scientifica SciCam Pro). Cells were recorded in whole-cell patch-clamp mode using pipettes pulled from standard-wall borosilicate capillaries using a (3.5-6 MOhm, DMZ Zeitz- Puller). Intracellular solution contained (in mM): 140 K-gluconate, 10 KCI, 10 HEPES, 4 Naphosphocreatine, 4 ATP-Mg, 0.4 GTP and biocytin (4 mg / mL) and was calibrated to pH 7.3 with KOH and an osmolality of 290-300 mOsm. A Multiclamp 700B amplifiers (Axon Instruments, CA) was used for whole-cell voltage clamp or current clamp recordings, together with a Digidata1550 (Molecular Devices) for digitization. Recordings were low pass filtered at a 10kHz using a Bessel filter and digitized at 50kHz. Series resistance was routinely compensated in voltage clamp, and recordings were excluded if access resistance exceeded 30MOhm.

[0313] Intrinsic electrophysiological properties’. L2 / 3 pyramidal cells were identified morphologically in hexb+ / - and hexb- / - transgenic mice. Input resistance was obtained from current traces evoked by a hyperpolarizing step (10 mV, 100 ms) and resting potential was determined in current clamp mode. Spiking profiles were recorded in current-clamp configuration (membrane potential was kept at -70 mV by passing a holding current) and the threshold current for spiking was assessed by successive current steps (starting at -150 pA and increased by 20 pA every sweep of 1 s duration). For characterizing action potential parameters, a previously developed pipeline was used.

[0314] Connectivity. Spontaneous EPSCs were sampled for 5 minutes, digitally filtered at 1 kHz, and detected offline. For analysis, a low-pass Butterworth filter was applied with a cut-off frequency of 500 Hz. The amplitude and area thresholds for detection were 4 pA and 50 pA.ms. All events were manually validated, and artifacts were discarded by visual inspection.

[0315] Statistical Analysis’. Data are presented as mean ± SEM. Normality was assessed using Shapiro- Wilk’s test, D’Agostino & Pearson omnibus test, and Kolmogorov-Smirnov’s test, at a significance level of 0.05. A distribution was considered as normal if all tests were passed. When a dataset did not satisfy normality criteria, nonparametric statistics were applied. Two-tailed Mann-Whitney’s test was used for single comparisons. For normal distributions, homoscedasticity was assessed using Ftest, at a significance level of 0.05. For homogeneous variances, two-tailed t test was used for single comparisons.

[0316] Gene expression analysis: RNA was isolated with the Arcturus Pico Pure RNA Isolation Kit (KIT0204, Life Technologies) according to the manufacturer’s protocol. Reverse transcription and real-time quantitative PCR analysis were performed using high capacity RNA-to-cDNA-Kit and Gene Expression Master Mix reagents (4387406, 4369510, Applied Biosystems) according to the manufacturer’s recommendations. The following TaqMan Gene Expression Assays were used: Actb (Mm01205647_g1), Gfap (Mm01253033_m1), Itgam (Mm00434455_m1), Syt1 (Mm00436858_m1), Plp1 (Mm01297210_m1), Hexb (Mm01282432_m1), Ccl5 (Mm01302427_m1), Cx3cl1 (Mm00436454_m1), 111 b (Mm00434228_m1), and 1118 (Mm004344226_m1). q PC Rs were run on a LightCycler 480 (Roche). Serum analysis: 100-200 pl of serum was analyzed for liver and renal parameters at Synlab.vet Augsburg. TNFa and IL-6 were analyzed using the TNFa and IL-6 ProQuantum Immunoassay Kits (A43656, A43658, Invitrogen) following the manufacturer’s instructions.

[0317] Statistics and Reproducibility: Statistical significance was determined using GraphPad Prism 10.2.2 software, p-values < 0.05 were considered statistically significant. All quantification experiments were performed in a blinded manner by assignment of unidentifiable numbers to mice, tissues and images for data acquisition and processing. Data labels and groups were only reinstated for statistical analysis. Quantification and imaging was not repeated following statistical analysis.

[0318] Ganglioside stimulation and slice culture were independently repeated twice with consistent results; representative data from one experiment are shown.

[0319] Experiments involving treatment of primary microglia with secretion inhibitors and Hex uptake assays in neuron and fibroblast cultures were also independently repeated twice with similar outcomes; data from both replicates were pooled and presented in the respective graphs.

[0320] Representative immunofluorescence / immunohistochemical micrographs are shown from multiple replicates.

[0321] REFERENCES

[0322] 1. Prinz M, Masuda T, Wheeler MA, Quintana FJ. Microglia and Central Nervous System- Associated Macrophages-From Origin to Disease Modulation. Annu Rev Immunol. 2021 Apr 26;39:251-277. doi: 10.1146 / annurev-immunol-093019-110159. Epub 2021 Feb 8. PMID: 33556248; PMCID: PMC8085109.

[0323] 2. Sango K, McDonald MP, Crawley JN, Mack ML, Tifft CJ, Skop E, Starr CM, Hoffmann A, Sandhoff K, Suzuki K, Praia RL. Mice lacking both subunits of lysosomal betahexosaminidase display gangliosidosis and mucopolysaccharidosis. Nat Genet. 1996 Nov;14(3):348-52. doi: 10.1038 / ng1196-348. PMID: 8896570.

[0324] 3. Wen J, Wang S, Guo R, Liu D. CSF1 R inhibitors are emerging immunotherapeutic drugs for cancer treatment. Eur J Med Chem. 2023 Jan 5;245(Pt 1):114884. doi: 10.1016 / j.ejmech.2022.114884. Epub 2022 Oct 29. PMID: 36335744.

[0325] 4. Fujiwara T, Yakoub MA, Chandler A, Christ AB, Yang G, Ouerfelli O, Rajasekhar VK, Yoshida A, Kondo H, Hata T, Tazawa H, Dogan Y, Moore MAS, Fujiwara T, Ozaki T, Purdue E, Healey JH. CSF1 / CSF1 R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor- Associated Macrophages and Stimulates T-cell Infiltration in the Sarcoma Microenvironment. Mol Cancer Ther. 2021 Aug;20(8):1388-1399. doi: 10.1158 / 1535-7163. MCT-20-0591 . Epub 2021 Jun 4. PMID: 34088832; PMCID: PMC9336538.

Claims

1. CLAIMS1 . A pharmaceutical combination comprising a. a colony-stimulating factor 1 receptor (CSF1 R) inhibitor and b. a bone marrow cell population for use in the treatment of central nervous system (CNS) diseases.

2. The pharmaceutical combination according to the preceding claim, wherein the CSF1 R inhibitor is an inhibitor of central nervous system (CNS)-associated myeloid cells.

3. The pharmaceutical combination according to any one of the preceding claims, wherein the CSF1 R inhibitor is an inhibitor of central nervous system (CNS)-associated macrophages (CAM) and / or microglia.

4. The pharmaceutical combination according to any one of the preceding claims, wherein the CSF1 R inhibitor depletes mutant and / or dysfunctional microglia and / or CNS-associated macrophages (CAM) in a central nervous system (CNS)-wide manner.

5. The pharmaceutical combination according to any one of the preceding claims, wherein the CSF1 R inhibitor inhibits genes associated with a central nervous system (CNS)-based immune response.

6. The pharmaceutical combination according to any one of the preceding claims, wherein the central nervous system (CNS)-associated myeloid cells carry a mutation in one or more genes associated with the CNS-based immune response, preferably in colony-stimulating factor 1 receptor (CSF1 R), ubiquitin-specific peptidase 18 (USP18), transmembrane immune signaling adaptor (TYROBP), triggering receptor expressed on myeloid cells 2 (TREM2), negative regulator of reactive oxygen species (NRROS), hexosaminidase A (HEXA), hexosaminidase B (HEXB), CD33, granulin (GRN), complement component 4 (C4), TNF receptor superfamily member 1A (TNFRSF1A), interferon regulatory factor s (IRF8), and / or purinergic receptor P2X 4 (P2X4R), more preferably CSF1 R, USP18, HEXB, and / or IRF8.

7. The pharmaceutical combination according to any one of the preceding claims, wherein the CSF1 R inhibitor is selected from BLZ945, PLX5622, PLX3397, ABT-869, SC-203877, OSI- 930, JNJ-40346527, CSF1 R-IN-1 , ABSK021 , ARRY-382, and / or AZD7507, preferably BLZ945, PLX5622, and / or PLX3397.

8. The pharmaceutical combination according to any one of the preceding claims, wherein the bone marrow cell population is autologous and / or allogeneic.

9. The pharmaceutical combination according to any one of the preceding claims, wherein the central nervous system (CNS) disease is a neurological condition and / or a mental disorder.

10. The pharmaceutical combination according to claim 9, wherein the central nervous system (CNS) disease is selected from lysosomal storage disease (LSD), microgliopathies, Parkinson's disease, Alzheimer's disease, frontotemporal dementia, multiple sclerosis, Rettsyndrome, autism spectrum disorder, depression, anxiety disorders, neuropathic pain, and / or schizophrenia.11 . The pharmaceutical combination according to claim 9, wherein the neurological condition is a lysosomal storage disease (LSD), preferably Sandhoff disease or Tay-Sachs disease.

12. The pharmaceutical combination according to claims 9 to 11 , wherein a patient is experiencing or suffering from one or more of symptoms, preferably cognitive and / or motoric symptoms, progressive nervous system deterioration, problems initiating and controlling muscles and / or movement, early blindness, seizures, and / or spasticity.

13. An ex vivo method for depleting dysfunctional and / or mutant microglia and / or CNS- associated macrophages (CAM), the method comprising a. providing a population of cells comprising myeloid cells, b. treating said cells with an CSF1 R inhibitor of central nervous system (CNS)- associated myeloid cells, thereby depleting dysfunctional and / or mutant microglia.

14. A cell population obtained according to the method of the preceding claim.

15. The cell population according to claim 14 for use in the treatment of central nervous system (CNS) diseases.

16. A pharmaceutical composition comprising the CSF1 R inhibitor according to claims 1 to 7 for use in the treatment or prevention of a medical condition induced by dysfunction of immune cells in a central nervous system (CNS), additionally comprising a pharmaceutically acceptable carrier.

17. The pharmaceutical composition for use according to the preceding claim, wherein the composition is administered intracerebrally, intrathecally, intravenously, or subcutaneously.

Images