Derya R. Shimshek

Recovery from peripheral nerve injury involves coordinated axonal and immune repair programs that promote tissue regeneration. However, the mechanisms regulating immune-mediated repair remain incompletely understood. Here, we demonstrate that glycoprotein non-metastatic melanoma protein B (GPNMB) is a key regulator of repair processes following peripheral nerve injury. In a mouse sciatic nerve crush model, loss of Gpnmb impaired functional recovery after injury. MRI and histological analyses revealed persistent myelin debris accumulation, defective debris clearance, and impaired macrophage lysosomal function within injured nerves. Mechanistic studies showed that GPNMB-deficient human iPSC-derived macrophages exhibited impaired lysosomal adaptation, reduced lysosomal fitness, and defective processing of myelin-associated cargo, resulting in inefficient degradation of injury-associated material. These defects were accompanied by impaired acquisition of repair-associated cellular programs. Together, our findings identify GPNMB as a critical regulator of lysosome-dependent repair programs during nerve regeneration and demonstrate that efficient GPNMB-dependent myelin processing is required for functional recovery following peripheral nerve injury. Graphical GPNMB enables efficient nerve regeneration after injury GPNMB promotes lysosomal adaptation in injury-responsive macrophages and microglia, enabling efficient myelin debris clearance and successful nerve regeneration. GPNMB coordinates injury-responsive myeloid and glial remodeling following peripheral nerve injury. After sciatic nerve crush, GPNMB is induced in macrophages, Schwann cells, and spinal cord microglia, where it promotes lysosomal adaptation and efficient myelin debris processing. By supporting reparative cellular states and sustained debris clearance, GPNMB enables axonal regeneration and functional recovery.

Age is a major determinant of demyelination and remyelination capacity. Here, we investigated age‑dependent brain responses to acute cuprizone exposure in female C57BL/6J mice using longitudinal multimodal MRI and histological analyses. Adult (6‑month‑old) and aged (18‑month‑old) mice received 0.2% cuprizone in the diet for 5 weeks, followed by 4 weeks of recovery on normal chow, with in vivo MRI performed at baseline, during intoxication, and throughout recovery. Magnetization transfer ratio (MTR) and T2‑weighted signal were used as MRI surrogates of myelin integrity and neuroinflammation, respectively, whereas T1‑weighted signal was assessed as a marker sensitive to oxidative stress. In adult mice, cuprizone induced robust and region‑specific pathology, including reduced MTR and increased T2‑weighted signal in the corpus callosum/external capsule, accompanied by microgliosis, astrocytosis, and increased APP‑positive dystrophic neurites. These abnormalities showed only partial recovery following cuprizone withdrawal. In contrast, cuprizone elicited little or no detectable change in these forebrain white‑matter regions in aged mice. Notably, the cerebellar nuclei remained susceptible at both ages, exhibiting persistent MTR decreases and T2‑weighted signal increases, corroborated by histological evidence of demyelination and gliosis. T1‑weighted signal increased broadly in adult, but not aged, mice during intoxication, consistent with cuprizone‑associated oxidative stress. Together, these findings demonstrate pronounced age‑ and region‑dependent effects in the cuprizone model and identify the cerebellar nuclei as a sensitive target of demyelination across the lifespan. Moreover, the non‑invasive nature of MRI enables efficient assessment of regional differences over time.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells and macrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases, such as Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knockout (TREM2-KO) and wild-type (WT) mice. The TREM2-sol mouse is a new model with selective elimination of plasma membrane TREM2 and a reduced expression of soluble TREM2. In the acute cuprizone model demyelination and remyelination events were reflected by a T2-weighted signal intensity change in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). In contrast to WT and TREM2-IPD, TREM2-sol and TREM2-KO showed an additional increase in MRI signal during the recovery phase. Histological analyses of TREM2-IPD animals revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 and displayed enhanced cytokine/chemokine levels in the brain. TREM2-sol and, to a much lesser extent, TREM2-KO, however, despite presenting reduced levels of some cytokines/chemokines, showed persistent microgliosis and astrocytosis during recovery, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia without plasma membrane TREM2 and only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function, as observed in bone marrow-derived macrophages, leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells andmacrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell-surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases like Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knock-out (TREM2-KO) and wildtype (WT) mice. In the acute cuprizone model consisting of a five-week intoxication by the copper chelator followed by a four-week recovery all genotypes showed demyelination at the peak of disease reflected by a T2-weighted signal intensity increase in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). During the recovery phase a partial signal reduction in WT and TREM2-IPD was observed, while TREM2-sol and TREM2-KO showed an additional increase in MRI signal in the EC. At week 9 histology revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 in TREM2-IPD animals, which also displayed enhanced cytokine/chemokine levels in the brain. Despite presenting reduced levels of some cytokines/chemokines, persistent microgliosis and astrocytosis at week 9 was likewise observed in TREM2-sol and to a much lesser extent in TREM2-KO, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia with only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts and monocytes/macrophages. In humans, homozygous loss-of-function mutation carriers exhibit bone cysts and fractures with an early-onset frontal lobe syndrome and progressive presenile dementia known as Nasu-Hakola. Neuropathological findings include myelin/neuronal loss and neuroinflammation. Heterozygous loss-of-function mutation in TREM2 has been associated with several neurodegenerative diseases like Alzheimer`s disease and Frontotemporal Dementia. We investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes by using different gene-modified animals expressing (cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knock-out (TREM2-KO) TREM2and wildtype (WT) mice in the cuprizone model.. In the acute cuprizone model all genotypes showed demyelination detected by T2-weighted signal intensity increase in Magnetic resonance imaging (MRI), most prominent in the external capsule (EC) and less in the corpus callosum. In the 4-week recovery phase a partial signal reduction in WT and TREM2-IPD was observed, while in TREM2-sol and TREM2-KO showed an increase in MRI signal in the EC. In TREM2-IPD histology revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1, which was also evidenced in enhanced cytokine/chemokine levels. This persistent microgliosis and astrocytosis was also observed in TREM2-sol, to a lesser extent in TREM2-KO during recovery with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. An increase in cytokines/chemokines was not evident. This was accompanied, specifically in the EC with no myelin recovery, appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic cuprizone model TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation leads to increased remyelination, whereas microglia without membrane-functional or complete loss of TREM2 results in a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology.

Background: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. Methods: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed. Results: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2–3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture’s genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions. Conclusion: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments.