David F. Meaney

Exogenous α-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death

Inclusions composed of α-synuclein (α-syn), i.e., Lewy bodies (LBs) and Lewy neurites (LNs), define synucleinopathies including Parkinsons disease (PD) and dementia with Lewy bodies (DLB). Here, we demonstrate that preformed fibrils generated from full-length and truncated recombinant α-syn enter primary neurons, probably by adsorptive-mediated endocytosis, and promote recruitment of soluble endogenous α-syn into insoluble PD-like LBs and LNs. Remarkably, endogenous α-syn was sufficient for formation of these aggregates, and overexpression of wild-type or mutant α-syn was not required. LN-like pathology first developed in axons and propagated to form LB-like inclusions in perikarya. Accumulation of pathologic α-syn led to selective decreases in synaptic proteins, progressive impairments in neuronal excitability and connectivity, and, eventually, neuron death. Thus, our data contribute important insights into the etiology and pathogenesis of PD-like α-syn inclusions and their impact on neuronal functions, and they provide a model for discovering therapeutics targeting pathologic α-syn-mediated neurodegeneration.

Diffuse axonal injury in head trauma.

Background:Diffuse axonal injury (DAI) is one of the most common and important pathologic features of traumatic brain injury (TBI). The susceptibility of axons to mechanical injury appears to be due to both their viscoelastic properties and their high organization in white matter tracts. Although axons are supple under normal conditions, they become brittle when exposed to rapid deformations associated with brain trauma. Accordingly, rapid stretch of axons can damage the axonal cytoskeleton resulting in a loss of elasticity and impairment of axoplasmic transport. Subsequent swelling of the axon occurs in discrete bulb formations or in elongated varicosities that accumulate transported proteins. Calcium entry into damaged axons is thought to initiate further damage by the activation of proteases. Ultimately, swollen axons may become disconnected and contribute to additional neuropathologic changes in brain tissue. DAI may largely account for the clinical manifestations of brain trauma. However, DAI is extremely difficult to detect noninvasively and is poorly defined as clinical syndrome. Conclusions:Future advancements in the diagnosis and treatment of DAI will be dependent on our collective understanding of injury biomechanics, temporal axonal pathophysiology, and its role in patient outcome.

Tissue-Level Thresholds for Axonal Damage in an Experimental Model of Central Nervous System White Matter Injury

In vivo, tissue-level, mechanical thresholds for axonal injury were determined by comparing morphological injury and electrophysiological impairment to estimated tissue strain in an in vivo model of axonal injury. Axonal injury was produced by dynamically stretching the right optic nerve of an adult male guinea pig to one of seven levels of ocular displacement (Nlevel = 10; Ntotal = 70). Morphological injury was detected with neurofilament immunohistochemical staining (NF68, SM132). Simultaneously, functional injury was determined by the magnitude of the latency shift of the N35 peak of the visual evoked potentials (VEPs) recorded before and after stretch. A companion set of in situ experiments (Nlevel = 5) was used to determine the empirical relationship between the applied ocular displacement and the magnitude of optic nerve stretch. Logistic regression analysis, combined with sensitivity and specificity measures and receiver operating characteristic (ROC) curves were used to predict strain thresholds for axonal injury. From this analysis, we determined three Lagrangian strain-based thresholds for morphological damage to white matter. The liberal threshold, intended to minimize the detection of false positives, was a strain of 0.34, and the conservative threshold strain that minimized the false negative rate was 0.14. The optimal threshold strain criterion that balanced the specificity and sensitivity measures was 0.21. Similar comparisons for electrophysiological impairment produced liberal, conservative, and optimal strain thresholds of 0.28, 0.13, and 0.18, respectively. With these threshold data, it is now possible to predict more accurately the conditions that cause axonal injury in human white matter.

Axonal Damage in Traumatic Brain Injury

Axonal damage is one of the most common and important pathologic features of traumatic brain injury. Severe diffuse axonal injury, resulting from inertial forces applied to the head, is associated with prolonged unconsciousness and poor outcome. The susceptibility of axons to mechanical injury appears to be due to both their viscoelastic properties and their highly organized structure in white matter tracts. Although axons are supple under normal conditions, they become brittle when exposed to rapid deformations associated with brain trauma. Accordingly, rapid stretch of axons can damage the axonal cytoskeleton, resulting in a loss of elasticity and impairment of axoplasmic transport. Subsequent swelling of the axon occurs in discrete bulb formations or in elongated varicosities that accumulate organelles. Calcium entry into damaged axons is thought to initiate further damage by the activation of proteases and the induction of mitochondrial swelling and dysfunction. Ultimately, swollen axons may become disconnected and contribute to additional neuropathologic changes in brain tissue. However, promising new therapies that reduce proteolytic activity or maintain mitochondrial integrity may attenuate progressive damage of injured axons following experimental brain trauma. Future advancements in the prevention and treatment of traumatic axonal injury will depend on our collective understanding of the relationship between the biomechanics and pathophysiology of various phases of axonal trauma.

Accumulation of Amyloid β and Tau and the Formation of Neurofilament Inclusions Following Diffuse Brain Injury in the Pig

Brain trauma in humans increases the risk for developing Alzheimer disease (AD) and may induce the acute formation of AD-like plaques containing amyloid beta (A beta). To further explore the potential link between brain trauma and neurodegeneration, we conducted neuropathological studies using a pig model of diffuse brain injury. Brain injury was induced in anesthetized animals via nonimpact head rotational acceleration of 110 degrees over 20 ms in the coronal plane (n = 15 injured, n = 3 noninjured). At 1, 3, 7, and 10 days post-trauma, control and injured animals were euthanized and immunohistochemical analysis was performed on brain sections using antibodies specific for A beta, beta-amyloid precursor protein (betaPP), tau, and neurofilament (NF) proteins. In addition to diffuse axonal pathology, we detected accumulation of A beta and tau that colocalized with immunoreactive betaPP and NF in damaged axons throughout the white matter in all injured animals at 3-10 days post-trauma. In a subset of brain injured animals, diffuse A beta-containing plaque-like profiles were found in both the gray and white matter, and accumulations of tau and NF rich inclusions were observed in neuronal perikarya. These results show that this pig model of diffuse brain injury is characterized by accumulations of proteins that also form pathological aggregates in AD and related neurodegenerative diseases.

Temporal window of vulnerability to repetitive experimental concussive brain injury.

OBJECTIVE:Repetitive concussive brain injury (CBI) is associated with cognitive alterations and increased risk of neurodegenerative disease. METHODS:To evaluate the temporal window during which the concussed brain remains vulnerable to a second concussion, anesthetized mice were subjected to either sham injury or single or repetitive CBI (either 3, 5, or 7 days apart) using a clinically relevant model of CBI. Cognitive, vestibular, and sensorimotor function (balance and coordination) were evaluated, and postmortem histological analyses were performed to detect neuronal degeneration, cytoskeletal proteolysis, and axonal injury. RESULTS:No cognitive deficits were observed in sham-injured animals or those concussed once. Mice subjected to a second concussion within 3 or 5 days exhibited significantly impaired cognitive function compared with either sham-injured animals (P < 0.05) or mice receiving a single concussion (P < 0.01). No cognitive deficits were observed when the interconcussion interval was extended to 7 days, suggestive of a transient vulnerability of the brain during the first 5 days after an initial concussion. Although all concussed mice showed transient motor deficits, vestibulomotor dysfunction was more pronounced in the group that sustained two concussions 3 days apart (P < 0.01 compared with all other groups). Although scattered degenerating neurons, evidence of cytoskeletal damage, and axonal injury were detected in selective brain regions between 72 hours and 1 week after injury in all animals sustaining a single concussion, the occurrence of a second concussion 3 days later resulted in significantly greater traumatic axonal injury (P < 0.05) than that resulting from a single CBI. CONCLUSION:These data suggest that a single concussion is associated with behavioral dysfunction and subcellular alterations that may contribute to a transiently vulnerable state during which a second concussion within 3 to 5 days can lead to exacerbated and more prolonged axonal damage and greater behavioral dysfunction.

mGluR5 stimulates gliotransmission in the nucleus accumbens

Although metabotropic glutamate receptor 5 (mGluR5) is essential for cocaine self-administration and drug-seeking behavior, there is limited knowledge of the cellular actions of this receptor in the nucleus accumbens (NAc). Although mGluR5 has the potential to regulate neurons directly, recent studies have shown the importance of mGluR5 in regulating Ca2+ signaling in astrocytes and, as a consequence, the Ca2+-dependent release of excitatory transmitters from these glia. In this study, we demonstrate that activation of mGluR5 induces Ca2+ oscillations in NAc astrocytes with the correlated appearance of NMDA receptor-dependent slow inward currents detected in medium spiny neurons (MSNs). Photolysis of caged Ca2+ loaded specifically into astrocytes evoked slow inward currents demonstrating that Ca2+ elevations in astrocytes are responsible for these excitatory events. Pharmacological evaluation of these glial-evoked NMDA currents shows that they are mediated by NR2B-containing NMDA receptors, whereas synaptic NMDA receptors rely on NR2A-containing receptors. Stimulation of glutamatergic afferents activates mGluR5-dependent astrocytic Ca2+ oscillations and gliotransmission that is sustained for minutes beyond the initial stimulus. Because gliotransmission is mediated by NMDA receptors, depolarized membrane potentials exhibited during up-states augment excitation provided by gliotransmission, which drives bursts of MSN action potentials. Because the predominant mGluR5-dependent action of glutamatergic afferents is to cause the sustained activation of astrocytes, which in turn excite MSNs through extrasynaptic NMDA receptors, our results raise the potential for gliotransmission being involved in prolonged mGluR5-dependent adaptation in the NAc.

Long-term accumulation of amyloid-β, β-secretase, presenilin-1, and caspase-3 in damaged axons following brain trauma

Plaques composed of amyloid β (Aβ) have been found within days following brain trauma in humans, similar to the hallmark plaque pathology of Alzheimers disease (AD). Here, we evaluated the potential source of this Aβ and long-term mechanisms that could lead to its production. Inertial brain injury was induced in pigs via head rotational acceleration of 110° over 20 ms in the coronal plane. Animals were euthanized at 3 hours, 3 days, 7 days, and 6 months post-injury. Immunohistochemistry and Western blot analyses of the brains were performed using antibodies specific for amyloid precursor protein (APP), Aβ peptides, β-site APP-cleaving enzyme (BACE), presenilin-1 (PS-1), caspase-3, and caspase-mediated cleavage of APP (CCA). Substantial co-accumulation for all of these factors was found in swollen axons at all time points up to 6 months following injury. Western blot analysis of injured brains confirmed a substantial increase in the protein levels of these factors, particularly in the white matter. These data suggest that impaired axonal transport due to trauma induces long-term pathological co-accumulation of APP with BACE, PS-1, and activated caspase. The abnormal concentration of these factors may lead to APP proteolysis and Aβ formation within the axonal membrane compartment.

Enhanced Astrocytic Ca2+ Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

Status epilepticus (SE), an unremitting seizure, is known to cause a variety of traumatic responses including delayed neuronal death and later cognitive decline. Although excitotoxicity has been implicated in this delayed process, the cellular mechanisms are unclear. Because our previous brain slice studies have shown that chemically induced epileptiform activity can lead to elevated astrocytic Ca2+ signaling and because these signals are able to induce the release of the excitotoxic transmitter glutamate from these glia, we asked whether astrocytes are activated during status epilepticus and whether they contribute to delayed neuronal death in vivo. Using two-photon microscopy in vivo, we show that status epilepticus enhances astrocytic Ca2+ signals for 3 d and that the period of elevated glial Ca2+ signaling is correlated with the period of delayed neuronal death. To ask whether astrocytes contribute to delayed neuronal death, we first administered antagonists which inhibit gliotransmission: MPEP [2-methyl-6-(phenylethynyl)pyridine], a metabotropic glutamate receptor 5 antagonist that blocks astrocytic Ca2+ signals in vivo, and ifenprodil, an NMDA receptor antagonist that reduces the actions of glial-derived glutamate. Administration of these antagonists after SE provided significant neuronal protection raising the potential for a glial contribution to neuronal death. To test this glial hypothesis directly, we loaded Ca2+ chelators selectively into astrocytes after status epilepticus. We demonstrate that the selective attenuation of glial Ca2+ signals leads to neuronal protection. These observations support neurotoxic roles for astrocytic gliotransmission in pathological conditions and identify this process as a novel therapeutic target.

Traumatic Axonal Injury Induces Proteolytic Cleavage of the Voltage-Gated Sodium Channels Modulated by Tetrodotoxin and Protease Inhibitors

We demonstrated previously that dynamic stretch injury of cultured axons induces structural changes and Ca2+ influx modulated by tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (NaChs). In the present study, we evaluated potential damage to the NaCh α-subunit, which can cause noninactivation of NaChs. In addition, we explored the effects of pre-injury and post-injury treatment with TTX and protease inhibition on proteolysis of the NaCh α-subunit and intra-axonal calcium levels ([Ca2+]i) over 60 min after trauma. After stretch injury, we found that [Ca2+]i continued to increase in untreated axons for at least 60 min. We also observed that the III-IV intra-axonal loop of the NaCh α-subunit was proteolyzed between 5 and 20 min after trauma. Pre-injury treatment of the axons with TTX completely abolished the posttraumatic increase in [Ca2+]i and proteolysis of the NaCh α-subunit. In addition, both pre-injury and post-injury inhibition of protease activity attenuated long-term increases in [Ca2+]i as well as mitigating degradation of the NaCh α-subunit. These results suggest a unique “feed-forward” deleterious process initiated by mechanical trauma of axons. Na+ influx through NaChs resulting from axonal deformation triggers initial increases in [Ca2+]i and subsequent proteolysis of the NaChα-subunit. In turn, degradation of the α-subunit promotes persistent elevations in [Ca2+]i, fueling additional pathologic changes. These observations may have important implications for developing therapeutic strategies for axonal trauma.