Brain injury drives optic glioma formation through neuron-glia signaling.

Tissue injury and tumorigenesis share many cellular and molecular features, including immune cell (T cells, monocytes) infiltration and inflammatory factor (cytokines, chemokines) elaboration. Their common pathobiology raises the intriguing possibility that brain injury could create a tissue microenvironment permissive for tumor formation. Leveraging several murine models of the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome and two experimental methods of brain injury, we demonstrate that both optic nerve crush and diffuse traumatic brain injury induce optic glioma (OPG) formation in mice harboring Nf1-deficient preneoplastic progenitors. We further elucidate the underlying molecular and cellular mechanisms, whereby glutamate released from damaged neurons stimulates IL-1ß release by oligodendrocytes to induce microglia expression of Ccl5, a growth factor critical for Nf1-OPG formation. Interruption of this cellular circuit using glutamate receptor, IL-1ß or Ccl5 inhibitors abrogates injury-induced glioma progression, thus establishing a causative relationship between injury and tumorigenesis.

Diffusion basis spectrum imaging detects subclinical traumatic optic neuropathy in a closed-head impact mouse model of traumatic brain injury.

Introduction: Traumatic optic neuropathy (TON) is the optic nerve injury secondary to brain trauma leading to visual impairment and vision loss. Current clinical visual function assessments often fail to detect TON due to slow disease progression and clinically silent lesions resulting in potentially delayed or missed treatment in patients with traumatic brain injury (TBI). Methods: Diffusion basis spectrum imaging (DBSI) is a novel imaging modality that can potentially fill this diagnostic gap. Twenty-two, 16-week-old, male mice were equally divided into a sham or TBI (induced by moderate Closed-Head Impact Model of Engineered Rotational Acceleration device) group. Briefly, mice were anesthetized with isoflurane (5% for 2.5 min followed by 2.5% maintenance during injury induction), had a helmet placed over the head, and were placed in a holder prior to a 2.1-joule impact. Serial visual acuity (VA) assessments, using the Virtual Optometry System, and DBSI scans were performed in both groups of mice. Immunohistochemistry (IHC) and histological analysis of optic nerves was also performed after in vivo MRI. Results: VA of the TBI mice showed unilateral or bilateral impairment. DBSI of the optic nerves exhibited bilateral involvement. IHC results of the optic nerves revealed axonal loss, myelin injury, axonal injury, and increased cellularity in the optic nerves of the TBI mice. Increased DBSI axon volume, decreased DBSI λ||, and elevated DBSI restricted fraction correlated with decreased SMI-312, decreased SMI-31, and increased DAPI density, respectively, suggesting that DBSI can detect coexisting pathologies in the optic nerves of TBI mice. Conclusion: DBSI provides an imaging modality capable of detecting subclinical changes of indirect TON in TBI mice.

Sex Differences in the Role of CNIH3 on Spatial Memory and Synaptic Plasticity.

BACKGROUND: CNIH3 is an AMPA receptor (AMPAR) auxiliary protein prominently expressed in the dorsal hippocampus (dHPC), a region that plays a critical role in spatial memory and synaptic plasticity. However, the effects of CNIH3 on AMPAR-dependent synaptic function and behavior have not been investigated. METHODS: We assessed a gain-of-function model of Cnih3 overexpression in the dHPC and generated and characterized a line of Cnih3-/- C57BL/6 mice. We assessed spatial memory through behavioral assays, protein levels of AMPAR subunits and synaptic proteins by immunoblotting, and long-term potentiation in electrophysiological recordings. We also utilized a super-resolution imaging workflow, SEQUIN (Synaptic Evaluation and Quantification by Imaging of Nanostructure), for analysis of nanoscale synaptic connectivity in the dHPC. RESULTS: Overexpression of Cnih3 in the dHPC improved short-term spatial memory in female mice but not in male mice. Cnih3-/- female mice exhibited weakened short-term spatial memory, reduced dHPC synapse density, enhanced expression of calcium-impermeable AMPAR (GluA2-containing) subunits in synaptosomes, and attenuated long-term potentiation maintenance compared with Cnih3+/+ control mice; Cnih3-/- males were unaffected. Further investigation revealed that deficiencies in spatial memory and changes in AMPAR composition and synaptic plasticity were most pronounced during the metestrus phase of the estrous cycle in female Cnih3-/- mice. CONCLUSIONS: This study identified a novel effect of sex and estrous on CNIH3's role in spatial memory and synaptic plasticity. Manipulation of CNIH3 unmasked sexually dimorphic effects on spatial memory, synaptic function, AMPAR composition, and hippocampal plasticity. These findings reinforce the importance of considering sex as a biological variable in studies of memory and hippocampal synaptic function.

Enhanced Multiplexing of Immunofluorescence Microscopy Using a Long-Stokes-Shift Fluorophore.

Immunofluorescence labeling and microscopy offer a highly specific means to visualize proteins or other molecular species in a sample by labeling target antigens with fluorescent probes. These fluorescent probes can then be visualized using a fluorescence microscope, allowing their relative spatial relationships to be determined. Due to spectral overlap of common fluorophores, however, it can be challenging to analyze more than three antigens in a single sample with standard imaging approaches. This article describes multiplexed labeling and imaging of four target antigens through the use of a long-Stokes-shift fluorophore-a fluorophore with an unusually large gap between its excitation and emission maxima-in tandem with three conventional fluorophores. This combination allows for multiplexed imaging of four antigens in a single sample with excellent spectral discrimination suitable for sensitive analyses using standard imaging hardware. Particular advantages of this approach are its flexibility in terms of target antigens and the lack of any specialized procedures, reagents, or equipment beyond the commercially available labeling reagent coupled to the long-Stokes-shift fluorophore. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Four-probe immunofluorescence labeling Basic Protocol 2: Four-probe immunofluorescence imaging.

Neuronal VCP loss of function recapitulates FTLD-TDP pathology.

The pathogenic mechanism by which dominant mutations in VCP cause multisystem proteinopathy (MSP), a rare neurodegenerative disease that presents as fronto-temporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), remains unclear. To explore this, we inactivate VCP in murine postnatal forebrain neurons (VCP conditional knockout [cKO]). VCP cKO mice have cortical brain atrophy, neuronal loss, autophago-lysosomal dysfunction, and TDP-43 inclusions resembling FTLD-TDP pathology. Conditional expression of a single disease-associated mutation, VCP-R155C, in a VCP null background similarly recapitulates features of VCP inactivation and FTLD-TDP, suggesting that this MSP mutation is hypomorphic. Comparison of transcriptomic and proteomic datasets from genetically defined patients with FTLD-TDP reveal that progranulin deficiency and VCP insufficiency result in similar profiles. These data identify a loss of VCP-dependent functions as a mediator of FTLD-TDP and reveal an unexpected biochemical similarity with progranulin deficiency.

Alpha-synuclein increases in rodent and human spinal cord injury and promotes inflammation and tissue loss.

Synucleinopathies are neurodegenerative diseases in which α-synuclein protein accumulates in neurons and glia. In these diseases, α-synuclein forms dense intracellular aggregates that are disease hallmarks and actively contribute to tissue pathology. Interestingly, many pathological mechanisms, including iron accumulation and lipid peroxidation, are shared between classical synucleinopathies such as Alzheimer's disease, Parkinson's disease and traumatic spinal cord injury (SCI). However, to date, no studies have determined if α-synuclein accumulation occurs after human SCI. To examine this, cross-sections from injured and non-injured human spinal cords were immunolabeled for α-synuclein. This showed robust α-synuclein accumulation in profiles resembling axons and astrocytes in tissue surrounding the injury, revealing that α-synuclein markedly aggregates in traumatically injured human spinal cords. We also detected significant iron deposition in the injury site, a known catalyst for α-synuclein aggregation. Next a rodent SCI model mimicking the histological features of human SCI revealed aggregates and structurally altered monomers of α-synuclein are present after SCI. To determine if α-synuclein exacerbates SCI pathology, α-synuclein knockout mice were tested. Compared to wild type mice, α-synuclein knockout mice had significantly more spared axons and neurons and lower pro-inflammatory mediators, macrophage accumulation, and iron deposition in the injured spinal cord. Interestingly, locomotor analysis revealed that α-synuclein may be essential for dopamine-mediated hindlimb function after SCI. Collectively, the marked upregulation and long-lasting accumulation of α-synuclein and iron suggests that SCI may fit within the family of synucleinopathies and offer new therapeutic targets for promoting neuron preservation and improving function after spinal trauma.

Acute Trem2 reduction triggers increased microglial phagocytosis, slowing amyloid deposition in mice.

Heterozygous genetic variants within the TREM2 gene show a strong association with increased Alzheimer's disease (AD) risk. Amyloid beta-depositing mouse models haploinsufficient or null for Trem2 have identified important relationships among TREM2, microglia, and AD pathology; however, results are challenging to interpret in the context of varying microglial phenotypes and disease progression. We hypothesized that acute Trem2 reduction may alter amyloid pathology and microglial responses independent of genetic Trem2 deletion in mouse models. We developed antisense oligonucleotides (ASOs) that potently but transiently lower Trem2 messenger RNA throughout the brain and administered them to APP/PS1 mice at varying stages of plaque pathology. Late-stage ASO-mediated Trem2 knockdown significantly reduced plaque deposition and attenuated microglial association around plaque deposits when evaluated 1 mo after ASO injection. Changes in microglial gene signatures 1 wk after ASO administration and phagocytosis measured in ASO-treated cells together indicate that microglia may be activated with short-term Trem2 reduction. These results suggest a time- and/or dose-dependent role for TREM2 in mediating plaque deposition and microglial responses in which loss of TREM2 function may be beneficial for microglial activation and plaque removal in an acute context.

Liver inflammation at the time of spinal cord injury enhances intraspinal pathology, liver injury, metabolic syndrome and locomotor deficits.

The current high obesity rates mean that neurological injuries are increasingly sustained on a background of systemic pathology, including liver inflammation, which likely has a negative impact on outcomes. Because obesity involves complex pathology, the effect of hepatic inflammation alone on neurological recovery is unknown. Thus, here we used a gain-of-function model to test if liver inflammation worsens outcome from spinal cord injury (SCI) in rats. Results show liver inflammation concomitant with SCI exacerbated intraspinal pathology and impaired locomotor recovery. Hepatic inflammation also potentiated SCI-induced non-alcoholic steatohepatitis (NASH), endotoxemia and insulin resistance. Circulating and cerebrospinal levels of the liver-derived protein Fetuin-A were higher in SCI rats with liver inflammation, and, when microinjected into intact spinal cords, Fetuin-A caused macrophage activation and neuron loss. Thus, liver inflammation functions as a disease modifying factor to impair recovery from SCI, and Fetuin-A is a potential neuropathological mediator. Since SCI alone induces acute liver inflammation, the liver may be a novel clinical target for improving recovery from SCI.

SEQUIN: An imaging and analysis platform for quantification and characterization of synaptic structures in mouse.

Synapses are crucial to brain function and frequent disease targets, but current analysis methods cannot report on individual synaptic components in situ or present barriers to widespread adoption. SEQUIN was developed to address this challenge. SEQUIN utilizes a widely available super-resolution platform in tandem with image processing and analysis to quantify synaptic loci over large regions of brain and characterize their molecular and nanostructural properties at the individual and population level. This protocol describes quantification of synaptic loci using SEQUIN. For additional details on the use and execution of this protocol, please refer to Sauerbeck et al. (2020).

Impact of TREM2R47H variant on tau pathology-induced gliosis and neurodegeneration.

Alzheimer's disease (AD) is characterized by plaques containing amyloid-ß (Aß) and neurofibrillary tangles composed of aggregated, hyperphosphorylated tau. Beyond tau and Aß, evidence suggests that microglia play an important role in AD pathogenesis. Rare variants in the microglia-expressed triggering receptor expressed on myeloid cells 2 (TREM2) gene increase AD risk 2- to 4-fold. It is likely that these TREM2 variants increase AD risk by decreasing the response of microglia to Aß and its local toxicity. However, neocortical Aß pathology occurs many years before neocortical tau pathology in AD. Thus, it will be important to understand the role of TREM2 in the context of tauopathy. We investigated the impact of the AD-associated TREM2 variant (R47H) on tau-mediated neuropathology in the PS19 mouse model of tauopathy. We assessed PS19 mice expressing human TREM2CV (common variant) or human TREM2R47H. PS19-TREM2R47H mice had significantly attenuated brain atrophy and synapse loss versus PS19-TREM2CV mice. Gene expression analyses and CD68 immunostaining revealed attenuated microglial reactivity in PS19-TREM2R47H versus PS19-TREM2CV mice. There was also a decrease in phagocytosis of postsynaptic elements by microglia expressing TREM2R47H in the PS19 mice and in human AD brains. These findings suggest that impaired TREM2 signaling reduces microglia-mediated neurodegeneration in the setting of tauopathy.

SEQUIN Multiscale Imaging of Mammalian Central Synapses Reveals Loss of Synaptic Connectivity Resulting from Diffuse Traumatic Brain Injury.

The brain's complex microconnectivity underlies its computational abilities and vulnerability to injury and disease. It has been challenging to illuminate the features of this synaptic network due to the small size and dense packing of its elements. Here, we describe a rapid, accessible super-resolution imaging and analysis workflow-SEQUIN-that quantifies central synapses in human tissue and animal models, characterizes their nanostructural and molecular features, and enables volumetric imaging of mesoscale synaptic networks without the production of large histological arrays. Using SEQUIN, we identify cortical synapse loss resulting from diffuse traumatic brain injury, a highly prevalent connectional disorder. Similar synapse loss is observed in three murine models of Alzheimer-related neurodegeneration, where SEQUIN mesoscale mapping identifies regional synaptic vulnerability. These results establish an easily implemented and robust nano-to-mesoscale synapse quantification and characterization method. They furthermore identify a shared mechanism-synaptopathy-between Alzheimer neurodegeneration and its best-established epigenetic risk factor, brain trauma.

Dietary Green Tea Extract Prior to Spinal Cord Injury Prevents Hepatic Iron Overload but Does Not Improve Chronic Hepatic and Spinal Cord Pathology in Rats.

Spinal cord injury (SCI) disrupts autonomic regulation of visceral organs. As a result, a leading cause of mortality in the SCI population is metabolic dysfunction, and an organ central to metabolic control is the liver. Our recent work showed that rodent SCI promotes Kupffer cell (hepatic macrophage) activation, pro-inflammatory cytokine expression, and liver steatosis. These are symptoms of nonalcoholic steatohepatitis (NASH), the hepatic manifestation of metabolic syndrome, and these pre-clinical data replicate aspects of post-SCI human metabolic dysfunction. Because metabolic profile is highly dependent on lifestyle, including diet, it is likely that lifestyle choices prior to injury influence metabolic and hepatic outcomes after SCI. Therefore, in this study we tested if a diet rich in green tea extract (GTE), a known hepatoprotective agent, that began 3 weeks before SCI and was maintained after injury, reduced indices of liver pathology or metabolic dysfunction. GTE treatment significantly reduced post-SCI hepatic iron accumulation and blunted circulating glucose elevation compared with control-diet rats. However, GTE pre-treatment did not prevent Kupffer cell activation, hepatic lipid accumulation, increased serum alanine transaminase, or circulating non-esterified fatty acids, which were all significantly increased 6 weeks post-injury. Spinal cord pathology also was unchanged by GTE. Thus, dietary GTE prior to and after SCI had only a minor hepatoprotective effect. In general, for optimal health of SCI individuals, it will be important for future studies to evaluate how other lifestyle choices made before or after SCI positively or negatively impact systemic and intraspinal outcomes and the overall metabolic health of SCI individuals.

Minimal Long-Term Neurobehavioral Impairments after Endovascular Perforation Subarachnoid Hemorrhage in Mice.

Cognitive deficits are among the most severe and pervasive consequences of aneurysmal subarachnoid hemorrhage (SAH). A critical step in developing therapies targeting such outcomes is the characterization of experimentally-tractable pre-clinical models that exhibit multi-domain neurobehavioral deficits similar to those afflicting humans. We therefore searched for neurobehavioral abnormalities following endovascular perforation induction of SAH in mice, a heavily-utilized model. We instituted a functional screen to manage variability in injury severity, then assessed acute functional deficits, as well as activity, anxiety-related behavior, learning and memory, socialization, and depressive-like behavior at sub-acute and chronic time points (up to 1 month post-injury). Animals in which SAH was induced exhibited reduced acute functional capacity and reduced general activity to 1 month post-injury. Tests of anxiety-related behavior including central area time in the elevated plus maze and thigmotaxis in the open field test revealed increased anxiety-like behavior at subacute and chronic time-points, respectively. Effect sizes for subacute and chronic neurobehavioral endpoints in other domains, however, were small. In combination with persistent variability, this led to non-significant effects of injury on all remaining neurobehavioral outcomes. These results suggest that, with the exception of anxiety-related behavior, alternate mouse models are required to effectively analyze cognitive outcomes after SAH.

Spinal cord injury causes chronic liver pathology in rats.

Traumatic spinal cord injury (SCI) causes major disruption to peripheral organ innervation and regulation. Relatively little work has investigated these post-SCI systemic changes, however, despite considerable evidence that multiple organ system dysfunction contributes to chronic impairments in health. Because metabolic dysfunction is common after SCI and the liver is a pivotal site for metabolic homeostasis, we sought to determine if liver pathology occurs as a result of SCI in a rat spinal contusion model. Histologic evidence showed excess lipid accumulation in the liver for at least 21 days post-injury after cervical or midthoracic SCI. Lipidomic analysis revealed an acute increase in hepatic ceramides as well as chronically elevated lactosylceramide. Post-SCI hepatic changes also included increased proinflammatory gene expression, including interleukin (IL)-1α, IL-1ß, chemokine ligand-2, and tumor necrosis factor-α mRNA. These were coincident with increased CD68+ macrophages in the liver through 21 days post-injury. Serum alanine transaminase, used clinically to detect liver damage, was significantly increased at 21 days post-injury, suggesting that early metabolic and inflammatory damage preceded overt liver pathology. Surprisingly, liver inflammation was even detected after lumbar SCI. Collectively, these results suggest that SCI produces chronic liver injury with symptoms strikingly similar to those of nonalcoholic steatohepatitis (fatty liver disease). These clinically significant hepatic changes after SCI are known to contribute to systemic inflammation, cardiovascular disease, and metabolic syndrome, all of which are more prevalent in persons with SCI. Targeting acute and prolonged hepatic pathology may improve recovery and reduce long-term complications after SCI.

Mitochondria, Amyloid ß, and Alzheimer's Disease.

Hypometabolism is a hallmark of Alzheimer's disease (AD) and implicates a mitochondrial role in the neuropathology associated with AD. Mitochondrial amyloid-beta (Aß) accumulation precedes extracellular Aß deposition. In addition to increasing oxidative stress, Aß has been shown to directly inhibit mitochondrial enzymes. Inhibition of mitochondrial enzymes as a result of oxidative damage or Aß interaction perpetuates oxidative stress and leads to a hypometabolic state. Additionally, Aß has also been shown to interact with cyclophilin D, a component of the mitochondrial permeability transition pore, which may promote cell death. Therefore, ample evidence exists indicating that the mitochondrion plays a vital role in the pathophysiology observed in AD.