Central and peripheral tau retention modulated by an anti-tau antibody.

Tau protein blood levels dependent on its distribution to peripheral organs and possible elimination from the body. Thus, the peripheral distribution of CSF-derived tau protein was explored, especially since there is a transition to blood-based biomarkers and the emerging idea that tau pathology may spread beyond brain. Near infrared fluorescence (NIRF) was mainly used to analyze tau (tau-NIRF) distribution after its intracisternal or intravenous injection. There was a striking uptake of blood- or CSF-derived tau-NIRF protein by the skeletal structures, liver, small intestine (duodenum), gall bladder, kidneys, urinary bladder, lymph nodes, heart, and spleen. In aging and in older APP/PS1 mice, tau uptake in regions, such as the brain, liver, and skeleton, was increased. In bone (femur) injected tau protein was associated with integrin-binding sialoprotein (IBSP), a major non-collagenous glycoprotein that is associated with mineralization. Tau-NIRF was cleared slowly from CSF via mainly across the cribriform plate, and cervical lymph nodes. In brain, some of the CSF injected tau protein was associated with NeuN-positive and PDGFRý-positive cells, which may explain its retention. The presence of tau in the bladders suggested excretion routes of tau. CSF anti-tau antibody increased CSF tau clearance, while blood anti-tau antibody decreased tau accumulation in the femur but not in liver, kidney, and spleen. Thus, the data show a body-wide distribution and retention of CSF-derived tau protein, which increased with aging and in older APP/PS1 mice. Further work is needed to elucidate the relevance of tau accumulation in each organ to tauopathy.

Uptake of severe acute respiratory syndrome coronavirus 2 spike protein mediated by angiotensin converting enzyme 2 and ganglioside in human cerebrovascular cells.

Introduction: There is clinical evidence of neurological manifestations in coronavirus disease-19 (COVID-19). However, it is unclear whether differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/spike protein (SP) uptake by cells of the cerebrovasculature contribute to significant viral uptake to cause these symptoms. Methods: Since the initial step in viral invasion is binding/uptake, we used fluorescently labeled wild type and mutant SARS-CoV-2/SP to study this process. Three cerebrovascular cell types were used (endothelial cells, pericytes, and vascular smooth muscle cells), in vitro. Results: There was differential SARS-CoV-2/SP uptake by these cell types. Endothelial cells had the least uptake, which may limit SARS-CoV-2 uptake into brain from blood. Uptake was time and concentration dependent, and mediated by angiotensin converting enzyme 2 receptor (ACE2), and ganglioside (mono-sialotetrahexasylganglioside, GM1) that is predominantly expressed in the central nervous system and the cerebrovasculature. SARS-CoV-2/SPs with mutation sites, N501Y, E484K, and D614G, as seen in variants of interest, were also differentially taken up by these cell types. There was greater uptake compared to that of the wild type SARS-CoV-2/SP, but neutralization with anti-ACE2 or anti-GM1 antibodies was less effective. Conclusion: The data suggested that in addition to ACE2, gangliosides are also an important entry point of SARS-CoV-2/SP into these cells. Since SARS-CoV-2/SP binding/uptake is the initial step in the viral penetration into cells, a longer exposure and higher titer are required for significant uptake into the normal brain. Gangliosides, including GM1, could be an additional potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.

Spike protein multiorgan tropism suppressed by antibodies targeting SARS-CoV-2.

While there is SARS-CoV-2 multiorgan tropism in severely infected COVID-19 patients, it's unclear if this occurs in healthy young individuals. In addition, for antibodies that target the spike protein (SP), it's unclear if these reduce SARS-CoV-2/SP multiorgan tropism equally. We used fluorescently labeled SP-NIRF to study viral behavior, using an in vivo dynamic imaging system and ex in vivo tissue analysis, in young mice. We found a SP body-wide biodistribution followed by a slow regional elimination, except for the liver, which showed an accumulation. SP uptake was highest for the lungs, and this was followed by kidney, heart and liver, but, unlike the choroid plexus, it was not detected in the brain parenchyma or CSF. Thus, the brain vascular barriers were effective in restricting the entry of SP into brain parenchyma in young healthy mice. While both anti-ACE2 and anti-SP antibodies suppressed SP biodistribution and organ uptake, anti-SP antibody was more effective. By extension, our data support the efficacy of these antibodies on SARS-CoV-2 multiorgan tropism, which could determine COVID-19 organ-specific outcomes.

SARS-CoV-2: is there neuroinvasion?

BACKGROUND: SARS-CoV-2, a coronavirus (CoV), is known to cause acute respiratory distress syndrome, and a number of non-respiratory complications, particularly in older male patients with prior health conditions, such as obesity, diabetes and hypertension. These prior health conditions are associated with vascular dysfunction, and the CoV disease 2019 (COVID-19) complications include multiorgan failure and neurological problems. While the main route of entry into the body is inhalation, this virus has been found in many tissues, including the choroid plexus and meningeal vessels, and in neurons and CSF. MAIN BODY: We reviewed SARS-CoV-2/COVID-19, ACE2 distribution and beneficial effects, the CNS vascular barriers, possible mechanisms by which the virus enters the brain, outlined prior health conditions (obesity, hypertension and diabetes), neurological COVID-19 manifestation and the aging cerebrovascualture. The overall aim is to provide the general reader with a breadth of information on this type of virus and the wide distribution of its main receptor so as to better understand the significance of neurological complications, uniqueness of the brain, and the pre-existing medical conditions that affect brain. The main issue is that there is no sound evidence for large flux of SARS-CoV-2 into brain, at present, compared to its invasion of the inhalation pathways. CONCLUSIONS: While SARS-CoV-2 is detected in brains from severely infected patients, it is unclear on how it gets there. There is no sound evidence of SARS-CoV-2 flux into brain to significantly contribute to the overall outcomes once the respiratory system is invaded by the virus. The consensus, based on the normal route of infection and presence of SARS-CoV-2 in severely infected patients, is that the olfactory mucosa is a possible route into brain. Studies are needed to demonstrate flux of SARS-CoV-2 into brain, and its replication in the parenchyma to demonstrate neuroinvasion. It is possible that the neurological manifestations of COVID-19 are a consequence of mainly cardio-respiratory distress and multiorgan failure. Understanding potential SARS-CoV-2 neuroinvasion pathways could help to better define the non-respiratory neurological manifestation of COVID-19.

Cerebrospinal fluid drainage kinetics across the cribriform plate are reduced with aging.

BACKGROUND: Continuous circulation and drainage of cerebrospinal fluid (CSF) are essential for the elimination of CSF-borne metabolic products and neuronal function. While multiple CSF drainage pathways have been identified, the significance of each to normal drainage and whether there are differential changes at CSF outflow regions in the aging brain are unclear. METHODS: Dynamic in vivo imaging of near infrared fluorescently-labeled albumin was used to simultaneously visualize the flow of CSF at outflow regions on the dorsal side (transcranial and -spinal) of the central nervous system. This was followed by kinetic analysis, which included the elimination rate constants for these regions. In addition, tracer distribution in ex vivo tissues were assessed, including the nasal/cribriform region, dorsal and ventral surfaces of the brain, spinal cord, cranial dura, skull base, optic and trigeminal nerves and cervical lymph nodes. RESULTS: Based on the in vivo data, there was evidence of CSF elimination, as determined by the rate of clearance, from the nasal route across the cribriform plate and spinal subarachnoid space, but not from the dorsal dural regions. Using ex vivo tissue samples, the presence of tracer was confirmed in the cribriform area and olfactory regions, around pial blood vessels, spinal subarachnoid space, spinal cord and cervical lymph nodes but not for the dorsal dura, skull base or the other cranial nerves. Also, ex vivo tissues showed retention of tracer along brain fissures and regions associated with cisterns on the brain surfaces, but not in the brain parenchyma. Aging reduced CSF elimination across the cribriform plate but not that from the spinal SAS nor retention on the brain surfaces. CONCLUSIONS: Collectively, these data show that the main CSF outflow sites were the nasal region across the cribriform plate and from the spinal regions in mice. In young adult mice, the contribution of the nasal and cribriform route to outflow was much higher than from the spinal regions. In older mice, the contribution of the nasal route to CSF outflow was reduced significantly but not for the spinal routes. This kinetic approach may have significance in determining early changes in CSF drainage in neurological disorder, age-related cognitive decline and brain diseases.

Glymphatic clearance controls state-dependent changes in brain lactate concentration.

Brain lactate concentration is higher during wakefulness than in sleep. However, it is unknown why arousal is linked to an increase in brain lactate and why lactate declines within minutes of sleep. Here, we show that the glymphatic system is responsible for state-dependent changes in brain lactate concentration. Suppression of glymphatic function via acetazolamide treatment, cisterna magna puncture, aquaporin 4 deletion, or changes in body position reduced the decline in brain lactate normally observed when awake mice transition into sleep or anesthesia. Concurrently, the same manipulations diminished accumulation of lactate in cervical, but not in inguinal lymph nodes when mice were anesthetized. Thus, our study suggests that brain lactate is an excellent biomarker of the sleep-wake cycle and increases further during sleep deprivation, because brain lactate is inversely correlated with glymphatic-lymphatic clearance. This analysis provides fundamental new insight into brain energy metabolism by demonstrating that glucose that is not fully oxidized can be exported as lactate via glymphatic-lymphatic fluid transport.

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation.

BACKGROUND: Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it's expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation. METHODS: We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student's t- test. RESULTS: We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2 > apoE3 > apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state. CONCLUSIONS: Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

Suppression of glymphatic fluid transport in a mouse model of Alzheimer's disease.

Glymphatic transport, defined as cerebrospinal fluid (CSF) peri-arterial inflow into brain, and interstitial fluid (ISF) clearance, is reduced in the aging brain. However, it is unclear whether glymphatic transport affects the distribution of soluble Aß in Alzheimer's disease (AD). In wild type mice, we show that Aß40 (fluorescently labeled Aß40 or unlabeled Aß40), was distributed from CSF to brain, via the peri-arterial space, and associated with neurons. In contrast, Aß42 was mostly restricted to the peri-arterial space due mainly to its greater propensity to oligomerize when compared to Aß40. Interestingly, pretreatment with Aß40 in the CSF, but not Aß42, reduced CSF transport into brain. In APP/PS1 mice, a model of AD, with and without extensive amyloid-ß deposits, glymphatic transport was reduced, due to the accumulation of toxic Aß species, such as soluble oligomers. CSF-derived Aß40 co-localizes with existing endogenous vascular and parenchymal amyloid-ß plaques, and thus, may contribute to the progression of both cerebral amyloid angiopathy and parenchymal Aß accumulation. Importantly, glymphatic failure preceded significant amyloid-ß deposits, and thus, may be an early biomarker of AD. By extension, restoring glymphatic inflow and ISF clearance are potential therapeutic targets to slow the onset and progression of AD.

The Effect of Body Posture on Brain Glymphatic Transport.

The glymphatic pathway expedites clearance of waste, including soluble amyloid ß (Aß) from the brain. Transport through this pathway is controlled by the brain's arousal level because, during sleep or anesthesia, the brain's interstitial space volume expands (compared with wakefulness), resulting in faster waste removal. Humans, as well as animals, exhibit different body postures during sleep, which may also affect waste removal. Therefore, not only the level of consciousness, but also body posture, might affect CSF-interstitial fluid (ISF) exchange efficiency. We used dynamic-contrast-enhanced MRI and kinetic modeling to quantify CSF-ISF exchange rates in anesthetized rodents' brains in supine, prone, or lateral positions. To validate the MRI data and to assess specifically the influence of body posture on clearance of Aß, we used fluorescence microscopy and radioactive tracers, respectively. The analysis showed that glymphatic transport was most efficient in the lateral position compared with the supine or prone positions. In the prone position, in which the rat's head was in the most upright position (mimicking posture during the awake state), transport was characterized by "retention" of the tracer, slower clearance, and more CSF efflux along larger caliber cervical vessels. The optical imaging and radiotracer studies confirmed that glymphatic transport and Aß clearance were superior in the lateral and supine positions. We propose that the most popular sleep posture (lateral) has evolved to optimize waste removal during sleep and that posture must be considered in diagnostic imaging procedures developed in the future to assess CSF-ISF transport in humans. SIGNIFICANCE STATEMENT: The rodent brain removes waste better during sleep or anesthesia compared with the awake state. Animals exhibit different body posture during the awake and sleep states, which might affect the brain's waste removal efficiency. We investigated the influence of body posture on brainwide transport of inert tracers of anesthetized rodents. The major finding of our study was that waste, including Aß, removal was most efficient in the lateral position (compared with the prone position), which mimics the natural resting/sleeping position of rodents. Although our finding awaits testing in humans, we speculate that the lateral position during sleep has advantage with regard to the removal of waste products including Aß, because clinical studies have shown that sleep drives Aß clearance from the brain.

Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism.

Metabolically, the brain is a highly active organ that relies almost exclusively on glucose as its energy source. According to the astrocyte-to-neuron lactate shuttle hypothesis, glucose is taken up by astrocytes and converted to lactate, which is then oxidized by neurons. Here we show, using two-photon imaging of a near-infrared 2-deoxyglucose analogue (2DG-IR), that glucose is taken up preferentially by neurons in awake behaving mice. Anaesthesia suppressed neuronal 2DG-IR uptake and sensory stimulation was associated with a sharp increase in neuronal, but not astrocytic, 2DG-IR uptake. Moreover, hexokinase, which catalyses the first enzymatic steps in glycolysis, was highly enriched in neurons compared with astrocytes, in mouse as well as in human cortex. These observations suggest that brain activity and neuronal glucose metabolism are directly linked, and identify the neuron as the principal locus of glucose uptake as visualized by functional brain imaging.

Biomarkers of traumatic injury are transported from brain to blood via the glymphatic system.

The nonspecific and variable presentation of traumatic brain injury (TBI) has motivated an intense search for blood-based biomarkers that can objectively predict the severity of injury. However, it is not known how cytosolic proteins released from traumatized brain tissue reach the peripheral blood. Here we show in a murine TBI model that CSF movement through the recently characterized glymphatic pathway transports biomarkers to blood via the cervical lymphatics. Clinically relevant manipulation of glymphatic activity, including sleep deprivation and cisternotomy, suppressed or eliminated TBI-induced increases in serum S100ß, GFAP, and neuron specific enolase. We conclude that routine TBI patient management may limit the clinical utility of blood-based biomarkers because their brain-to-blood transport depends on glymphatic activity.

Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury.

Traumatic brain injury (TBI) is an established risk factor for the early development of dementia, including Alzheimer's disease, and the post-traumatic brain frequently exhibits neurofibrillary tangles comprised of aggregates of the protein tau. We have recently defined a brain-wide network of paravascular channels, termed the "glymphatic" pathway, along which CSF moves into and through the brain parenchyma, facilitating the clearance of interstitial solutes, including amyloid-ß, from the brain. Here we demonstrate in mice that extracellular tau is cleared from the brain along these paravascular pathways. After TBI, glymphatic pathway function was reduced by ∼60%, with this impairment persisting for at least 1 month post injury. Genetic knock-out of the gene encoding the astroglial water channel aquaporin-4, which is importantly involved in paravascular interstitial solute clearance, exacerbated glymphatic pathway dysfunction after TBI and promoted the development of neurofibrillary pathology and neurodegeneration in the post-traumatic brain. These findings suggest that chronic impairment of glymphatic pathway function after TBI may be a key factor that renders the post-traumatic brain vulnerable to tau aggregation and the onset of neurodegeneration.

Impairment of paravascular clearance pathways in the aging brain.

OBJECTIVE: In the brain, protein waste removal is partly performed by paravascular pathways that facilitate convective exchange of water and soluble contents between cerebrospinal fluid (CSF) and interstitial fluid (ISF). Several lines of evidence suggest that bulk flow drainage via the glymphatic system is driven by cerebrovascular pulsation, and is dependent on astroglial water channels that line paravascular CSF pathways. The objective of this study was to evaluate whether the efficiency of CSF-ISF exchange and interstitial solute clearance is impaired in the aging brain. METHODS: CSF-ISF exchange was evaluated by in vivo and ex vivo fluorescence microscopy and interstitial solute clearance was evaluated by radiotracer clearance assays in young (2-3 months), middle-aged (10-12 months), and old (18-20 months) wild-type mice. The relationship between age-related changes in the expression of the astrocytic water channel aquaporin-4 (AQP4) and changes in glymphatic pathway function was evaluated by immunofluorescence. RESULTS: Advancing age was associated with a dramatic decline in the efficiency of exchange between the subarachnoid CSF and the brain parenchyma. Relative to the young, clearance of intraparenchymally injected amyloid-ß was impaired by 40% in the old mice. A 27% reduction in the vessel wall pulsatility of intracortical arterioles and widespread loss of perivascular AQP4 polarization along the penetrating arteries accompanied the decline in CSF-ISF exchange. INTERPRETATION: We propose that impaired glymphatic clearance contributes to cognitive decline among the elderly and may represent a novel therapeutic target for the treatment of neurodegenerative diseases associated with accumulation of misfolded protein aggregates.

The spectrum of neurobehavioral sequelae after repetitive mild traumatic brain injury: a novel mouse model of chronic traumatic encephalopathy.

There has been an increased focus on the neurological sequelae of repetitive mild traumatic brain injury (TBI), particularly neurodegenerative syndromes, such as chronic traumatic encephalopathy (CTE); however, no animal model exists that captures the behavioral spectrum of this phenomenon. We sought to develop an animal model of CTE. Our novel model is a modification and fusion of two of the most popular models of TBI and allows for controlled closed-head impacts to unanesthetized mice. Two-hundred and eighty 12-week-old mice were divided into control, single mild TBI (mTBI), and repetitive mTBI groups. Repetitive mTBI mice received six concussive impacts daily for 7 days. Behavior was assessed at various time points. Neurological Severity Score (NSS) was computed and vestibulomotor function tested with the wire grip test (WGT). Cognitive function was assessed with the Morris water maze (MWM), anxiety/risk-taking behavior with the elevated plus maze, and depression-like behavior with the forced swim/tail suspension tests. Sleep electroencephalogram/electromyography studies were performed at 1 month. NSS was elevated, compared to controls, in both TBI groups and improved over time. Repetitive mTBI mice demonstrated transient vestibulomotor deficits on WGT. Repetitive mTBI mice also demonstrated deficits in MWM testing. Both mTBI groups demonstrated increased anxiety at 2 weeks, but repetitive mTBI mice developed increased risk-taking behaviors at 1 month that persist at 6 months. Repetitive mTBI mice exhibit depression-like behavior at 1 month. Both groups demonstrate sleep disturbances. We describe the neurological sequelae of repetitive mTBI in a novel mouse model, which resemble several of the neuropsychiatric behaviors observed clinically in patients sustaining repetitive mild head injury.

The pathophysiology underlying repetitive mild traumatic brain injury in a novel mouse model of chronic traumatic encephalopathy.

BACKGROUND: An animal model of chronic traumatic encephalopathy (CTE) is essential for further understanding the pathophysiological link between repetitive head injury and the development of chronic neurodegenerative disease. We previously described a model of repetitive mild traumatic brain injury (mTBI) in mice that encapsulates the neurobehavioral spectrum characteristic of patients with CTE. We aimed to study the pathophysiological mechanisms underlying this animal model. METHODS: Our previously described model allows for controlled, closed head impacts to unanesthetized mice. Briefly, 12-week-old mice were divided into three groups: Control, single, and repetitive mTBI. Repetitive mTBI mice received six concussive impacts daily, for 7 days. Mice were then subsequently sacrificed for macro- and micro-histopathologic analysis at 7 days, 1 month, and 6 months after the last TBI received. Brain sections were immunostained for glial fibrillary acidic protein (GFAP) for astrocytes, CD68 for activated microglia, and AT8 for phosphorylated tau protein. RESULTS: Brains from single and repetitive mTBI mice lacked macroscopic tissue damage at all time-points. Single mTBI resulted in an acute rea ctive astrocytosis at 7 days and increased phospho-tau immunoreactivity that was present acutely and at 1 month, but was not persistent at 6 months. Repetitive mTBI resulted in a more marked neuroinflammatory response, with persistent and widespread astrogliosis and microglial activation, as well as significantly elevated phospho-tau immunoreactivity to 6-months. CONCLUSIONS: The neuropathological findings in this new model of repetitive mTBI resemble some of the histopathological hallmarks of CTE, including increased astrogliosis, microglial activation, and hyperphosphorylated tau protein accumulation.