Lipofuscin is an autofluorescent (AF) pigment formed by lipids and misfolded proteins, which accumulates in postmitotic cells with advanced age. Here, we immunophenotyped microglia in the brain of old C57BL/6 mice (>18 months old) and demonstrate that in comparison to young mice, one-third of old microglia are AF, characterized by profound changes in lipid and iron content, phagocytic activity, and oxidative stress. Pharmacological depletion of microglia in old mice eliminated the AF microglia following repopulation and reversed microglial dysfunction. Age-related neurological deficits and neurodegeneration after traumatic brain injury (TBI) were attenuated in old mice lacking AF microglia. Furthermore, increased phagocytic activity, lysosomal burden, and lipid accumulation in microglia persisted for up to 1 year after TBI, were modified by APOE4 genotype, and chronically driven by phagocyte-mediated oxidative stress. Thus, AF may reflect a pathological state in aging microglia associated with increased phagocytosis of neurons and myelin and inflammatory neurodegeneration that can be further accelerated by TBI.

Extracellular vesicles (EVs) have been implicated mechanistically in the pathobiology of neurodegenerative disorders, including central nervous system injury. However, the role of EVs in spinal cord injury (SCI) has received limited attention to date. Moreover, technical limitations related to EV isolation and characterization methods can lead to misleading or contradictory findings. Here, we examined changes in plasma EVs after mouse SCI at multiple timepoints (1d, 3d, 7d, 14d) using complementary measurement techniques. Plasma EVs isolated by ultracentrifugation (UC) were decreased at 1d post-injury, as shown by nanoparticle tracking analysis (NTA), and paralleled an overall reduction in total plasma extracellular nanoparticles. Western blot (WB) analysis of UC-derived plasma EVs revealed increased expression of the tetraspanin exosome marker, CD81, between 1d and 7d post-injury. To substantiate these findings, we performed interferometric and fluorescence imaging of single, tetraspanin EVs captured directly from plasma with ExoView®. Consistent with WB, we observed significantly increased plasma CD81+ EV count and cargo at 1d post-injury. The majority of these tetraspanin EVs were smaller than 50 nm based on interferometry and were insufficiently resolved by flow cytometry-based detection. At the injury site, there was enhanced expression of EV biogenesis proteins that were also detected in EVs directly isolated from spinal cord tissue by WB. Surface expression of tetraspanins CD9 and CD63 increased in multiple cell types at the injury site; however, astrocyte CD81 expression uniquely decreased, as demonstrated by flow cytometry. UC-isolated plasma EV microRNA cargo was also significantly altered at 1d post-injury with changes similar to that reported in EVs released by astrocytes after inflammatory stimulation. When injected into the lateral ventricle, plasma EVs from SCI mice increased both pro- and anti-inflammatory gene as well as reactive astrocyte gene expression in the brain cortex. These studies provide the first detailed characterization of plasma EV dynamics after SCI and suggest that plasma EVs may be involved in posttraumatic brain inflammation.

Evaluation of the chronic effects of spinal cord injury (SCI) has long focused on sensorimotor deficits, neuropathic pain, bladder/bowel dysfunction, loss of sexual function, and emotional distress. Although not well appreciated clinically, SCI can cause cognitive impairment including deficits in learning and memory, executive function, attention, and processing speed; it also commonly leads to depression. Recent large-scale longitudinal population-based studies indicate that patients with isolated SCI (without concurrent brain injury) are at a high risk of dementia associated with substantial cognitive impairments. Yet, little basic research has addressed potential mechanisms for cognitive impairment and depression after injury. In addition to contributing to disability in their own right, these changes can adversely affect rehabilitation and recovery and reduce quality of life. Here, we review clinical and experimental work on the complex and varied responses in the brain following SCI. We also discuss potential mechanisms responsible for these less well-examined, important SCI consequences. In addition, we outline the existing and developing therapeutic options aimed at reducing SCI-induced brain neuroinflammation and post-injury cognitive and emotional impairments.

Traumatic brain injury (TBI) can cause progressive neurodegeneration, sustained neuroinflammation and chronic neurological dysfunction. Few experimental studies have explored the long-term neurobehavioral and functional cellular changes beyond several months. The present study examined the effects of a single moderate-level TBI on functional outcome 8 months after injury. Male C57BL/6 mice were subjected to controlled cortical impact injury and followed for changes in motor performance, learning and memory, as well as depressive-like and social behavior. We also used a novel flow cytometry approach to assess cellular functions in freshly isolated neurons and microglia from the injured tissue. There were marked and diverse, sustained neurobehavioral changes in injured mice. Compared to sham controls, chronic TBI mice showed long-term deficits in gait dynamics, nest building, spatial working memory and recognition memory. The tail suspension, forced swim, and sucrose consumption tests showed a marked depressive-like phenotype that was associated with impaired sociability. At the cellular level, there were lower numbers of Thy1+Tuj1+ neurons and higher numbers of activated CD45loCD11b+ microglia. Functionally, both neurons and microglia exhibited significantly higher levels of oxidative stress after injury. Microglia exhibited chronic deficits in phagocytosis of E. coli bacteria, and increased uptake of myelin and dying neurons. Living neurons showed decreased expression of synaptophysin and postsynaptic density (PSD)-95, along with greater numbers of microtubule-associated protein light chain 3 (LC3)-positive autophagosomes and increased mitochondrial mass that suggest dysregulation of autophagy. In summary, the late neurobehavioral changes found after murine TBI are similar to those found chronically after moderate-severe human head injury. Importantly, such changes are associated with microglial dysfunction and changes in neuronal activity.

Whereas human spinal cord injury (SCI) is more common in men, the prevalence is growing in women. However, little is known about the effect of biological sex on brain dysfunction and injury mechanisms. To model the highest per capita rate of injury (ages between 16–30 years old) in humans, in the present study, young adult or a young/middle-aged male and female C57BL/6 mice were subjected to moderate contusion SCI. When mice were injured at 10–12-week-old, transcriptomic analysis of inflammation-related genes and flow cytometry revealed a more aggressive neuroinflammatory profile in male than females following 3 d SCI, ostensibly driven by sex-specific changes myeloid cell function rather than cell number. Female mice were generally more active at baseline, as evidenced by greater distance traveled in the open field. After SCI, female mice had more favorable locomotor function than male animals. At 13 weeks post-injury, male mice showed poor performance in cognitive and depressive-like behavioral tests, while injured female mice showed fewer deficits in these tasks. However, when injured at 6 months old followed by 8 months post-injury, male mice had considerably less inflammatory activation compared with female animals despite having similar or worse outcomes in affective, cognitive, and motor tasks. Collectively, these findings indicate that sex differences in functional outcome after SCI are associated with the age at onset of injury, as well as disrupted neuroinflammation not only at the site of injury but also in remote brain regions. Thus, biological sex should be considered when designing new therapeutic agents.

Autophagy is a cellular catabolic pathway generally thought to be neuroprotective. However, autophagy and in particular its upstream regulator, the ULK1 kinase, can also promote axonal degeneration. We examined the role and the mechanisms of autophagy in axonal degeneration using a mouse model of contusive spinal cord injury (SCI). Consistent with activation of autophagy during axonal degeneration following SCI, autophagosome marker LC3, ULK1 kinase, and ULK1 target, phospho-ATG13, accumulated in the axonal bulbs and injured axons. SARM1, a TIR NADase with a pivotal role in axonal degeneration, colocalized with ULK1 within 1 h after SCI, suggesting possible interaction between autophagy and SARM1-mediated axonal degeneration. In our in vitro experiments, inhibition of autophagy, including Ulk1 knockdown and ULK1 inhibitor, attenuated neurite fragmentation and reduced accumulation of SARM1 puncta in neurites of primary cortical neurons subjected to glutamate excitotoxicity. Immunoprecipitation data demonstrated that ULK1 physically interacted with SARM1 in vitro and in vivo and that SAM domains of SARM1 were necessary for ULK1–SARM1 complex formation. Consistent with a role in regulation of axonal degeneration, in primary cortical neurons ULK1–SARM1 interaction increased upon neurite damage. Supporting a role for autophagy and ULK1 in regulation of SARM1 in axonal degeneration in vivo, axonal ULK1 activation and accumulation of SARM1 were both decreased after SCI in Becn1 ^+/− autophagy hypomorph mice compared to wild-type (WT) controls. These findings suggest a regulatory crosstalk between autophagy and axonal degeneration pathways, which is mediated through ULK1–SARM1 interaction and contributes to the ability of SARM1 to accumulate in injured axons.