To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type–specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.

Compartmentalized inflammation is a key driver of multiple sclerosis (MS) progression, but the mechanisms sustaining its persistence remain unclear. A hallmark of this persistent and slowly evolving inflammatory process is chronic active MS lesions. We generated a high-resolution, single-cell molecular and spatial atlas of such lesions by combining single-nucleus RNA sequencing (snRNA-seq) with multiplexed error-robust fluorescence in situ hybridization (MERFISH). Within lesion rims, we identified CD8⁺ T cell niches associated with inflamed microglia displaying an interferon response and upregulated lipid metabolism. To investigate their function, we deleted ATP-binding cassette transporters A1 and G1 (ABCA1/G1) in the microglia of mice with experimental autoimmune encephalomyelitis (EAE), which increased the formation of lipid-storing phagocytes that amplified inflammation. Moreover, pharmacologically targeting sterol metabolism mitigated foam cell formation and inflammatory demyelination in EAE. Thus, our high-resolution map of immune niches in chronic active MS lesions identifies a role for lipid-storing, dysfunctional microglia in persistent neuroinflammation.

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD- GRN ). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD- GRN , namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD- GRN –associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)–derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD- GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD- GRN and potentially other CNS disorders.

The velocity of axonal impulse propagation is facilitated by myelination and axonal diameters. Both parameters are frequently impaired in peripheral nerve disorders, but it is not known if the diameters of myelinated axons affect the liability to injury or the efficiency of functional recovery. Mice lacking the adaxonal myelin protein chemokine‐like factor‐like MARVEL‐transmembrane domain‐containing family member‐6 (CMTM6) specifically from Schwann cells (SCs) display appropriate myelination but increased diameters of peripheral axons. Here we subjected Cmtm6‐cKo mice as a model of enlarged axonal diameters to a mild sciatic nerve compression injury that causes temporarily reduced axonal diameters but otherwise comparatively moderate pathology of the axon/myelin‐unit. Notably, both of these pathological features were worsened in Cmtm6‐cKo compared to genotype‐control mice early post‐injury. The increase of axonal diameters caused by CMTM6‐deficiency thus does not override their injury‐dependent decrease. Accordingly, we did not detect signs of improved regeneration or functional recovery after nerve compression in Cmtm6‐cKo mice; depleting CMTM6 in SCs is thus not a promising strategy toward enhanced recovery after nerve injury. Conversely, the exacerbated axonal damage in Cmtm6‐cKo nerves early post‐injury coincided with both enhanced immune response including foamy macrophages and SCs and transiently reduced grip strength. Our observations support the concept that larger peripheral axons are particularly susceptible toward mechanical trauma.

In the central nervous system, myelin formed around nerve axons by oligodendrocyte enables efficient conduction of action potentials and maintains axonal homeostasis. Therefore, in demyelinating diseases, such as multiple sclerosis (MS), axonal degeneration and loss, particularly in small‐diameter axons, are observed. Teneurin‐4 deficient (Ten‐4 ‐/‐) mice exhibit severe dysmyelination of small‐diameter axons, but survive for over a year. Here, we aimed to elucidate the mechanism of the survival of small‐diameter axons without myelin using Ten‐4 ‐/‐ mice. An immunohistochemical analysis showed abnormally diffused and intense staining of neurofilament. Further, the immunohistochemical signal of nonphosphorylated neurofilament and amyloid precursor protein was higher in Ten‐4 ‐/‐ mice. An electron microscopy analysis unexpectedly revealed that small‐diameter axons in Ten‐4 ‐/‐ mice survived at the age of 1 year, despite the absence of compact myelin. To elucidate the molecular mechanism, we performed a proteomics analysis in the Ten‐4 ‐/‐ tissue by mass spectrometry. The result indicated that four of the seven 14‐3‐3 isoforms were highly expressed in Ten‐4 ‐/‐ small‐diameter axons. Finally, when the function of 14‐3‐3s in Ten‐4 ‐/‐ mice was inhibited by 14‐3‐3 inhibitors, BV02 and difopein, the progression of small‐diameter axon damage was observed. Altogether, 14‐3‐3s are among the pro‐survival factors in dysmyelinated small‐diameter axons. Our findings may be useful for better understanding the pathogenesis of demyelinating diseases, such as MS, as well as for the development of new treatments.

Dysfunctional immune responses impair remyelination and contribute to neurodegeneration. While immune and metabolic responses to acute myelin injury are well characterized, myeloid cell adaptations to chronic myelin damage remain poorly understood. Here we performed a comparative analysis of genetic and toxic mouse models of acute and chronic demyelination, which identified a distinct microglial state associated with chronic demyelination, characterized by sustained pro-inflammatory activation, lysosomal lipid sequestration and reduced lipid droplet formation. Transcriptomics profiling further revealed a metabolic program marked by upregulated cholesterol synthesis and diminished sterol efflux. Treatment with a desmosterol mimetic possessing 24-dehydrocholesterol reductase inhibitor activity reversed this microglial state, attenuated pro-inflammatory signaling (including C-C Motif Chemokine Ligand 4 expression), reduced myelin damage and improved motor performance in a mouse model of leukodystrophy. Our findings uncover a pro-inflammatory microglial state characterized by lysosomal dysfunction and increased myelin reactivity, highlighting a novel therapeutic target for chronic demyelinating disorders.