DOCK2-deficiency causes defects in anti-viral T cell responses and impaired control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, analysis of these in patients is complicated by their treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. We also uncovered a critical, cell intrinsic role of DOCK2 in the priming of anti-viral CD8+ T cells and in particular their initial expansion, despite apparently normal early activation of these cells. When this defect was overcome by priming in vitro, DOCK2-deficient CD8+ T cells were surprisingly protective against HSV-1-disease, albeit not as effectively as wild type cells. These results shed light on a cellular deficiency that is likely to impact anti-viral immunity in DOCK2-deficient patients.

DOCK2-deficiency causes defects in anti-viral T cell responses and poor control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, patients with these diseases are by definition rare. In addition, any analysis is complicated by treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. Further, we found that they have a critical, cell intrinsic role of DOCK2 in the clonal expansion of anti-viral CD8+ T cells despite normal early activation of these cells. Finally, while the major deficiency is in clonal expansion, the ability of primed and expanded DOCK2-deficient CD8+ T cells to protect against HSV-1-infection is also compromised. These results provide a contributing cause for the frequent and devastating viral infections seen in DOCK2-deficient patients and improve our understanding of anti-viral CD8+ T cell immunity.

The case against secondary epileptogenesis.

Secondary epileptogenesis is a theory that hypothesizes that uncontrolled seizures in people with epilepsy lead to the development of new sites of seizure onset. This process has often been cited when people experience a new seizure type after a period of poor seizure control. The theory proposes that repeated seizures induce changes in regions of the brain that are regularly recruited into the seizure. These hypothetical changes can then lead to a new, independent seizure onset zone. The concept is based on a number of clinical observations which secondary epileptogenesis could explain. However there are alternative explanations from the clinic as well as from the laboratory that call the process into question. In this review some of the observations that have been used to support the theory will be reviewed, and the many counterarguments will be presented. At this time there is little evidence to support secondary epileptogenesis and much to refute it.

Noninvasive disconnection of targeted neuronal circuitry sparing axons of passage and nonneuronal cells.

OBJECTIVE: Surgery can be highly effective for the treatment of medically intractable, neurological disorders, such as drug-resistant focal epilepsy. However, despite its benefits, surgery remains substantially underutilized due to both surgical concerns and nonsurgical impediments. In this work, the authors characterized a noninvasive, nonablative strategy to focally destroy neurons in the brain parenchyma with the goal of limiting collateral damage to nontarget structures, such as axons of passage. METHODS: Low-intensity MR-guided focused ultrasound (MRgFUS), together with intravenous microbubbles, was used to open the blood-brain barrier (BBB) in a transient and focal manner in rats. The period of BBB opening was exploited to focally deliver to the brain parenchyma a systemically administered neurotoxin (quinolinic acid) that is well tolerated peripherally and otherwise impermeable to the BBB. RESULTS: Focal neuronal loss was observed in targeted areas of BBB opening, including brain regions that are prime objectives for epilepsy surgery. Notably, other structures in the area of neuronal loss, including axons of passage, glial cells, vasculature, and the ventricular wall, were spared with this procedure. CONCLUSIONS: These findings identify a noninvasive, nonablative approach capable of disconnecting neural circuitry while limiting the neuropathological consequences that attend other surgical procedures. Moreover, this strategy allows conformal targeting, which could enhance the precision and expand the treatment envelope for treating irregularly shaped surgical objectives located in difficult-to-reach sites. Finally, if this strategy translates to the clinic, the noninvasive nature and specificity of the procedure could positively influence both physician referrals for and patient confidence in surgery for medically intractable neurological disorders.

Chronic limbic epilepsy models for therapy discovery: Protocols to improve efficiency.

OBJECTIVE: There have been recommendations to improve therapy discovery for epilepsy by incorporating chronic epilepsy models into the preclinical process, but unpredictable seizures and difficulties in maintaining drug levels over prolonged periods have been obstacles to using these animals. We report new protocols in which drugs are administered through a new chronic gastric tube to rats with higher seizure frequencies to minimize these obstacles. METHODS: Adult rats with spontaneous limbic seizures following an episode of limbic status epilepticus induced by electrical hippocampal stimulation were monitored with long-term video- electroencephalography (EEG). Animals with a predetermined baseline seizure frequency received an intragastric tube for drug administration. Carbamazepine, levetiracetam, phenobarbital, and phenytoin were tested with either an acute protocol (an increasing single dose every other day for a maximum of three doses) or with a chronic protocol (multiple administrations of one dose for a week). Drug levels were obtained to correlate the effect with the level. RESULTS: With the acute protocol, all four drugs induced a clear dose-related response. Similar dose-related responses were seen following the week-long dosing protocol for carbamazepine, phenobarbital, and phenytoin, and these responses were associated with drug levels that were in the human therapeutic range. The response to chronic levetiracetam was much less robust. The gastric tube route of administration was well tolerated over a number of months. SIGNIFICANCE: Using rats with stable, higher seizure frequencies made it possible to identify the potential of a drug to suppress seizures in a realistic model of epilepsy with drug levels that are similar to those of human therapeutic levels. The acute protocol provided a full dose response in 1 week. The chronic administration protocol further differentiated drugs that may be effective long term. The gastric tube facilitates a less stressful, humane, and consistent administration of multiple doses.

Non-invasive, neurotoxic surgery reduces seizures in a rat model of temporal lobe epilepsy.

Surgery can be highly effective for treating certain cases of drug resistant epilepsy. The current study tested a novel, non-invasive, surgical strategy for treating seizures in a rat model of temporal lobe epilepsy. The surgical approach uses magnetic resonance-guided, low-intensity focused ultrasound (MRgFUS) in combination with intravenous microbubbles to open the blood-brain barrier (BBB) in a transient and focal manner. During the period of BBB opening, a systemically administered neurotoxin (Quinolinic Acid: QA) that is normally impermeable to the BBB gains access to a targeted area in the brain, destroying neurons where the BBB has been opened. This strategy is termed Precise Intracerebral Non-invasive Guided Surgery (PING). Spontaneous recurrent seizures induced by pilocarpine were monitored behaviorally prior to and after PING or under control conditions. Seizure frequency in untreated animals or animals treated with MRgFUS without QA exhibited expected seizure rate fluctuations frequencies between the monitoring periods. In contrast, animals treated with PING targeting the intermediate-temporal aspect of the hippocampus exhibited substantial reductions in seizure frequency, with convulsive seizures being eliminated entirely in two animals. These findings suggest that PING could provide a useful alternative to invasive surgical interventions for treating drug resistant epilepsy, and perhaps for treating other neurological disorders in which aberrant neural circuitries play a role.

NINJ1 mediates plasma membrane rupture during lytic cell death.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

Recurrent limbic seizures do not cause hippocampal neuronal loss: A prolonged laboratory study.

PURPOSE: It remains controversial whether neuronal damage and synaptic reorganization found in some forms of epilepsy are the result of an initial injury and potentially contributory to the epileptic condition or are the cumulative affect of repeated seizures. A number of reports of human and animal pathology suggest that at least some neuronal loss precedes the onset of seizures, but there is debate over whether there is further damage over time from intermittent seizures. In support of this latter hypothesis are MRI studies in people that show reduced hippocampal volumes and cortical thickness with longer durations of the disease. In this study we addressed the question of neuronal loss from intermittent seizures using kindled rats (no initial injury) and rats with limbic epilepsy (initial injury). METHODS: Supragranular mossy fiber sprouting, hippocampal neuronal densities, and subfield area measurements were determined in rats with chronic limbic epilepsy (CLE) that developed following an episode of limbic status epilepticus (n = 25), in kindled rats (n = 15), and in age matched controls (n = 20). To determine whether age or seizure frequency played a role in the changes, CLE and kindled rats were further classified by seizure frequency (low/high) and the duration of the seizure disorder (young/old). RESULTS: Overall there was no evidence for progressive neuronal loss from recurrent seizures. Compared with control and kindled rats, CLE animals showed increased mossy fiber sprouting, decreased neuronal numbers in multiple regions and regional atrophy. In CLE, but not kindled rats: 1) Higher seizure frequency was associated with greater mossy fiber sprouting and granule cell dispersion; and 2) greater age with seizures was associated with decreased hilar densities, and increased hilar areas. There was no evidence for progressive neuronal loss, even with more than 1000 seizures. CONCLUSION: These findings suggest that the neuronal loss associated with limbic epilepsy precedes the onset of the seizures and is not a consequence of recurrent seizures. However, intermittent seizures do cause other structural changes in the brain, the functional consequences of which are unclear.

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry.

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic. Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The role of EEG in patients with suspected epilepsy.

Despite the advances in imaging, EEG remains a critical test for the diagnosis of epilepsy. Not only can it confirm the diagnosis, but it can also clarify the type of epilepsy. There are many different types of EEG recordings depending on duration, the presence of video, and inpatient or outpatient setting, each with its pros and cons. Interictal epileptiform abnormalities are very specific to epilepsy, but they can be over-interpreted by inexperienced readers. In addition to diagnosis of epilepsy, EEG also has a role in the decision to discontinue treatment in seizure-free patients, and in assessing critically ill patients for possible status epilepticus and encephalopathies. EEG reports should be relatively standardized and clear to the clinician who requested the EEG.

Effects of Non-invasive, Targeted, Neuronal Lesions on Seizures in a Mouse Model of Temporal Lobe Epilepsy.

Surgery to treat drug-resistant epilepsy can be quite effective but remains substantially underutilized. A pilot study was undertaken to test the feasibility of using a non-invasive, non-ablative, approach to produce focal neuronal loss to treat seizures in a rodent model of temporal lobe epilepsy. In this study, spontaneous, recurrent seizures were established in a mouse model of pilocarpine-induced status epilepticus. After post-status epilepticus stabilization, baseline behavioral seizures were monitored for 30 d. Non-invasive opening of the blood-brain barrier targeting the hippocampus was then produced by using magnetic resonance-guided, low-intensity focused ultrasound, through which a neurotoxin (quinolinic acid) administered intraperitoneally gained access to the brain parenchyma to produce focal neuronal loss. Behavioral seizures were then monitored for 30 d after this procedure, and brains were subsequently prepared for histologic analysis of the sites of neuronal loss. The average frequency of behavioral seizures in all animals (n = 11) was reduced by 21.2%. Histologic analyses along the longitudinal axis of the hippocampus revealed that most of the animals (n = 8) exhibited neuronal loss located primarily in the intermediate aspect of the hippocampus, while sparing the septal aspect. Two other animals with damage to the intermediate hippocampus also exhibited prominent bilateral damage to the septal aspect of the hippocampus. A final animal had negligible neuronal loss overall. Notably, the site of neuronal loss along the longitudinal axis of the hippocampus influenced seizure outcomes. Animals that did not have bilateral damage to the septal hippocampus displayed a mean decrease in seizure frequency of 27.7%, while those with bilateral damage to the septal hippocampus actually increased seizure frequency by 18.7%. The animal without neuronal loss exhibited an increase in seizure frequency of 19.6%. The findings indicate an overall decrease in seizure frequency in treated animals. And, the site of neuronal loss along the longitudinal axis of the hippocampus appears to play a key role in reducing seizure activity. These pilot data are promising, and they encourage additional and more comprehensive studies examining the effects of targeted, non-invasive, neuronal lesions for the treatment of epilepsy.

Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

IRF2 transcriptionally induces GSDMD expression for pyroptosis.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1ß (IL-1ß) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N-ethyl-N-nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2-deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1ß secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.

Inhibition of T-Type calcium channels in mEC layer II stellate neurons reduces neuronal hyperexcitability associated with epilepsy.

Temporal lobe epilepsy (TLE) is a form of adult epilepsy involving the entorhinal cortex (EC). Layer II neurons of the medial EC (mEC) are spared and become hyperexcitable in TLE. Studies have suggested a role for T-type calcium channels (T-type Ca2+ channels) in facilitating increases in neuronal activity associated with TLE within the hippocampus. We sought to determine if T-type Ca2+ channels play a role in facilitating neuronal hyperexcitability of layer II mEC stellate neurons in TLE. TLE was induced in rats by electrical stimulation of the hippocampus to induce status epilepticus (SE). Brain slices were prepared from rats exhibiting spontaneous seizures and compared with age-matched control rats. Action potentials (APs) were evoked either by current injection steps or via presynaptic stimulation of mEC deep layers. The selective T-type Ca2+ channel antagonist, TTA-P2 (1 µM), was applied to determine the role of T-type Ca2+ channels in maintaining neuronal excitability. Quantitative PCR techniques were used to assess T-type Ca2+ channel isoform mRNA levels within the mEC layer II. TLE mEC layer II stellate neurons were hyperexcitable compared to control neurons, evoking a higher frequency of APs and generating bursts of APs when synaptically stimulated. TTA-P2 (1 µM) reduced firing frequencies in TLE and control neurons and reduced AP burst firing in TLE stellate neurons. TTA-P2 had little effect on synaptically evoked AP's in control neurons. TTA-P2 also inhibited rebound APs evoked in TLE neurons to a greater degree than in control neurons. TLE tissue had almost a 3-fold increase in Cav3.1 mRNA compared to controls. Cav3.2 or Cav3.3 levels were unchanged. These findings support a role for T-type Ca2+ channel in establishing neuronal hyperexcitability of mEC layer II stellate neurons in TLE. Increased expression of Cav3.1 may be important for establishing neuronal hyperexcitability of mEC layer II neurons in TLE.

Modelling traumatic brain injury and posttraumatic epilepsy in rodents.

Posttraumatic epilepsy (PTE) is one of the most debilitating and understudied consequences of traumatic brain injury (TBI). It is challenging to study the effects, underlying pathophysiology, biomarkers, and treatment of TBI and PTE purely in human patients for a number of reasons. Rodent models can complement human PTE studies as they allow for the rigorous investigation into the causal relationship between TBI and PTE, the pathophysiological mechanisms of PTE, the validation and implementation of PTE biomarkers, and the assessment of PTE treatments, in a tightly controlled, time- and cost-efficient manner in experimental subjects known to be experiencing epileptogenic processes. This article will review several common rodent models of TBI and/or PTE, including their use in previous studies and discuss their relative strengths, limitations, and avenues for future research to advance our understanding and treatment of PTE.

Testing Different Combinations of Acoustic Pressure and Doses of Quinolinic Acid for Induction of Focal Neuron Loss in Mice Using Transcranial Low-Intensity Focused Ultrasound.

The goal of this study was to test different combinations of acoustic pressure and doses of quinolinic acid (QA) for producing a focal neuronal lesion in the murine hippocampus without causing unwanted damage to adjacent brain structures. Sixty male CD-1 mice were divided into 12 groups that underwent magnetic resonance-guided focused ultrasound at high (0.67 MPa), medium (0.5 MPa) and low (0.33 MPa) acoustic peak negative pressures and received QA at high (0.012 mmol), medium (0.006 mmol) and low (0.003 mmol) dosages. Neuronal loss occurred only when magnetic resonance-guided focused ultrasound with adequate acoustic power (0.67 or 0.5 MPa) was combined with QA. The animals subjected to the highest acoustic power had larger lesions than those treated with medium acoustic power, but two mice had evidence of bleeding. When the intermediate acoustic power was used, medium and high dosages of QA produced lesions larger than those produced by the low dosage.

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.

Genetic resistance to kindling associated with alterations in circuit function.

How a seizure spreads from a focal onset zone to other regions of the brain is not well understood, and animal studies suggest that there is a genetic influence. To understand how genetic factors may influence seizure spread, we examined whether the kindling resistance of WAG/Rij rats, which are slow to develop kindled motor seizures, is independent of the site of seizure induction and thus a global phenomenon, or whether it is circuit specific. We compared the kindling rates (number of stimulations to induce kindled motor seizures) of WAG/Rij rats to the rates of kindling in Sprague Dawley rats. Both groups underwent a standard hippocampal kindling protocol and a separate group was kindled from the medial dorsal nucleus of the thalamus, a site that has been previously demonstrated to result in the very rapid development of motor seizures. To examine whether there were differences in the interaction in a circuit involved with the motor seizures, evoked responses were obtained from the prefrontal cortex following stimulation of the subiculum or medial dorsal thalamic nucleus. The WAG/Rij rats once again demonstrated resistance to kindling in the hippocampus, but both strains kindled rapidly from the medial dorsal nucleus. In the WAG/Rij rats there was also a reduction in the duration of the afterdischarge in the frontal cortex during hippocampal stimulation, but there was no reduction during thalamic kindling. The prefrontal cortex evoked responses were reduced following stimulation of the subiculum in the WAG/Rij rats, but the evoked responses to thalamic stimulation were the same in both strains. These findings suggest that there are genetic influences in the strength of the input from the subiculum to the prefrontal cortex in WAG/Rij rats that could explain the resistance to limbic kindling because of reduced excitatory drive onto a key target region.