Faculty Bibliography

Background: To be effective, prophylactic HIV vaccines must elicit antibodies (Abs) against the virus envelope (Env). Although HIV Env is renowned for its variability, it also contains regions that are conserved, albeit poorly immunogenic. To direct the Ab response toward the more conserved Env sites, we utilized immune complex vaccines made of the Env protein gp120 or gp140 and monoclonal Abs (mAbs) against different gp120 epitopes. We previously demonstrated the ability of gp120/mAb immune complexes to enhance the elicitation of V3 Abs; however, Ab response to other regions, especially the V1V2 domain recently identified as an important target for protective Abs against HIV, has not been studied; neither have immune complex vaccines with non-B-subtype Env.
Method(s): This study compared immunogenicity of subtypes B (JRFL), C (CN54), and CRF-01.AE (A244) Env in complex with selected gp120-specific mAbs in mice.
Result(s): Immunization with the complexes elicited comparable serum IgG titers against Env, but a marked skewing toward V1V2 or V3 was evident and dependent on the Env strain and the specificity of the mAb used to form the complexes. Compared with gp120JRFL, immunization with gp120JRFL complexed with CD4bs or V1V2 mAbs, but not with C2 or V3 mAbs, elicited greater IgG titers against V3 from subtypes A, B, and C. Epitope mapping revealed a shift toward a more conserved site in the V3 crown. However, the complexes did not enhance V1V2 Ab response, and the elicited V1V2 Abs were not cross-reactive. This profile contrasts with Ab responses to gp140CN54/mAb and gp120A244/mAb complexes. Notably, gp120A244/ mAb complexes induced higher levels of V1V2 Abs, while stimulating weak or strain-specific V3 Abs. Along with altered immunogenicity, allosteric and antigenic changes were detected on these complexes, indicating that mAb interaction induces alterations on the Env surface that modify its immunogenic property.
Conclusion(s): Immune complex vaccines may be useful to shape Ab responses toward Env sites of interest

Neurotrophins play critical roles in the survival, maintenance and death of neurons. In particular, proneurotrophins have been shown to mediate cell death following brain injury induced by status epilepticus (SE) in rats. Previous studies have shown that pilocarpine-induced seizures lead to increased levels of proNGF, which binds to the p75NTR-sortilin receptor complex to elicit apoptosis. A screen to identify compounds that block proNGF binding and uptake into cells expressing p75 and sortilin identified lithium citrate as a potential inhibitor of proNGF and p75NTR-mediated cell death. In this study, we demonstrate that low, submicromolar doses of lithium citrate effectively inhibited proNGF-induced cell death in cultured neurons and protected hippocampal neurons following pilocarpine-induced SE in vivo. We analyzed specific mechanisms by which lithium citrate afforded neuroprotection and determined that lithium citrate prevented the association and internalization of the p75NTR-sortilin receptor complex. Our results demonstrate a novel mechanism by which low-dose treatments of lithium citrate are effective in attenuating p75NTR-mediated cell death in vitro and in vivo.

Background: The main component of the amyloid deposited in the brain of Alzheimer's disease patients is beta-amyloid (Abeta), a proteolytic product of the amyloid beta precursor protein (APP). Mature APP undergoes proteolytic cleavage by alpha- and beta-secretases to produce C-terminal fragments (APP-CTFs). beta-APP-CTF is a neurotoxic protein that is also the source of Abeta following cleavage by gamma-secretase. It was previously shown that amyloidogenic APP processing mainly occurs in endosomes and that exosomes contain APP, APP-CTFs, a minute fraction of Abeta, and the secretases involved in APP metabolism, but the exosomal contribution to amyloid pathology remains unknown. We have investigated whether APP processing occurs in the exosomal pathway. Methods: Exosomes were isolated from postmortem human and mouse brains, and from the culture media of human fibroblasts and of the neuroblastoma cell line SH-SY5Y. The content of APP, APP metabolites and APP secretases in exosomes was analysed by Western blot and compared with the content in the brain or cell homogenates. Results: We found that exosomes isolated from human and mouse brains as well as exosomes secreted by cells in vitro are enriched in APP-CTFs. All three APP secretases were detected in the exosome preparations and interestingly, beta-secretase 1 (BACE1) and the mature form of the -secretase ADAM10 were also enriched in exosomes, whereas the gamma-secretase subunit Nicastrin was not. Our data also show that exosomal beta- and alpha- secretases are active, based on the observation of continuous generation of APP-CTFs in isolated exosomes. Summary/Conclusion: Our data show that APP processing continues in exosomes following their release into the extracellular space from the endosomal multivesicular bodies, implicating exosomes as carriers and generation sites of the neurotoxic beta-APP-CTF and an extracellular source of Abeta. Given the stability of exosomes, this may propagate amyloid pathogenicity throughout the brain

Background: The apolipoprotein E (APOE) gene codes for the brain's primary cholesterol carrier protein. In both humans and humanized APOE mice the Alzheimer's disease-risk APOE 4 allele (APOE4) alters the number and size of neuronal endosomes, a pathology common to several neurodegenerative disorders, including Alzheimer's disease. Given that exosomes derive from the endosomal system, we investigated the impact of APOE4 on brain-derived exosomes. Methods: Extracellular vesicles (EV) were isolated from brain tissue of neuropathologically normal humans and of APOE targeted-replacement mice at 6, 12 and 18 months of age. Antibodies against TSG101 and ALIX were used to identify the exosome population within these samples. Protein, mRNA and lipid analyses were performed on both EV and whole-brain samples. Results: We found lower exosome levels in the brains of neuropathologically normal human APOE4 carriers compared to individuals homozygous for the risk-neutral 3 allele (APOE3). In APOE4 compared with APOE3 mice, brain exosome levels were lower in an age-dependent manner: lower levels were observed at 12 and 18 but not at 6 months of age. Protein and mRNA expressions of the exosome pathway regulators TSG101 and Rab35 were also lower in APOE4 compared with APOE3 mouse brains at 12 months of age, arguing for decreased exosome biosynthesis and secretion, respectively, from the endosomal pathway. Cholesterol and ganglioside levels were higher in brain exosomes isolated from 12-month-old APOE4 compared with APOE3 mice. Summary/Conclusion: Our findings show an APOE4-driven downregulation of brain exosome biosynthesis and release that is associated with altered lipid homeostasis. Failure to maintain proper functioning of the interdependent endosomal-exosomal pathways during aging, which is essential for diverse homeostatic and catabolic cellular processes, is likely to contribute to neuronal vulnerability in neurodegenerative disorders, including Alzheimer's disease

Background: Dysfunction of the neuronal endosomal pathway is a characteristic of down syndrome (DS) and Alzheimer's disease (AD) and of carriers of the AD-risk apolipoprotein E 4 allele (APOE4). We hypothesized that the efficient release of endosomal material via exosomes into the extracellular space, as observed in the brains of DS patients and a mouse model of the disease and by DS fibroblasts, is necessary for a neuron to prevent accumulation of endosomal contents. Conversely, APOE4-driven downregulation of exosome release in the brains of APOE4 human carriers and APOE4 targeted-replacement mice appears to contribute to endosomal pathology. We investigated in vitro the interrelationship between the endosomal and exosomal pathways. Methods: Fibroblasts from DS patients and age-matched controls were transfected with CD63 siRNA or negative control siRNA. Level of exosomal secretion was studied by western blot analysis, and number and area of endosomes by immunohistochemistry. Results: Knockdown of the tetraspanin CD63, a regulator of exosome biogenesis, diminished exosome release by DS fibroblasts but not by control cells. CD63 knockdown did not affect endosomal morphology in control cells, but the number and total area occupied by endosomes was greater in DS fibroblasts in which CD63 expression was reduced. Summary/Conclusion: In neurodegenerative disorders with endosomallysosomal dysfunction, exosome secretion serves as a disposal mechanism for potentially toxic materials that are abnormally accumulated in endosomal compartments. Conversely, APOE4-driven downregulation of brain exosome biosynthesis and release contributes to endosomal pathology. Failure to maintain proper functioning of the interdependent endosomal-exosomal pathways during aging likely contributes to neuron degeneration and our findings argue that exosome production plays a central role maintaining homeostatic function of the endosomal-lysosomal system

A remarkable example of maladaptive plasticity is the development of epilepsy after a brain insult or injury to a normal animal or human. A structure that is considered central to the development of this type of epilepsy is the dentate gyrus (DG), because it is normally a relatively inhibited structure and its quiescence is thought to reduce hippocampal seizure activity. This characteristic of the DG is also considered to be important for normal hippocampal-dependent cognitive functions. It has been suggested that the brain insults which cause epilepsy do so because they cause the DG to be more easily activated. One type of brain insult that is commonly used is induction of severe seizures (status epilepticus; SE) by systemic injection of a convulsant drug. Here we describe an alteration in the DG after this type of experimental SE that may contribute to chronic seizures that has not been described before: large folds or gyri that develop in the DG by 1 month after SE. Large gyri appeared to increase network excitability because epileptiform discharges recorded in hippocampal slices after SE were longer in duration when recorded inside gyri relative to locations outside gyri. Large gyri may also increase excitability because immature adult-born neurons accumulated at the base of gyri with time after SE, and previous studies have suggested that abnormalities in adult-born DG neurons promote seizures after SE. In summary, large gyri after SE are a common finding in adult rats, show increased excitability, and are associated with the development of an abnormal spatial distribution of adult-born neurons. Together these alterations may contribute to chronic seizures and associated cognitive comorbidities after SE.

Epilepsy is a neurologic condition which often occurs with other neurologic and psychiatric disorders. The relation between epilepsy and these conditions is complex. Some population-based studies have identified a bidirectional relation, whereby not only patients with epilepsy are at increased risk of suffering from some of these neurologic and psychiatric disorders (migraine, stroke, dementia, autism, depression, anxiety disorders, Attention deficit hyperactivity disorder (ADHD), and psychosis), but also patients with these conditions are at increased risk of suffering from epilepsy. The existence of common pathogenic mechanisms has been postulated as a potential explanation of this phenomenon. To reassess the relationships between neurological and psychiatric conditions in general, and specifically autism, depression, Alzheimer's disease, schizophrenia, and epilepsy, a recent meeting brought together basic researchers and clinician scientists entitled "Epilepsy as a Network Disorder." This was the fourth in a series of conferences, the "Fourth International Halifax Conference and Retreat". This manuscript summarizes the proceedings on potential relations between Epilepsy on the one hand and autism and depression on the other. A companion manuscript provides a summary of the proceedings about the relation between epilepsy and Alzheimer's disease and schizophrenia, closed by the role of translational research in clarifying these relationships. The review of the topics in these two manuscripts will provide a better understanding of the mechanisms operant in some of the common neurologic and psychiatric comorbidities of epilepsy.

The major objective of preclinical translational epilepsy research is to advance laboratory findings toward clinical application by testing potential treatments in animal models of seizures and epilepsy. Recently there has been a focus on the failure of preclinical discoveries to translate reliably, or even to be reproduced in different laboratories. One potential cause is a lack of standardization in preclinical data collection. The resulting difficulties in comparing data across studies have led to high cost and missed opportunity, which in turn impede clinical trials and advances in medical care. Preclinical epilepsy research has successfully brought numerous antiseizure treatments into the clinical practice, yet the unmet clinical needs have prompted the reconsideration of research strategies to optimize epilepsy therapy development. In the field of clinical epilepsy there have been successful steps to improve such problems, such as generation of common data elements (CDEs) and case report forms (CRFs and standards of data collection and reporting) by a team of leaders in the field. Therefore, the Translational Task Force was appointed by the International League Against Epilepsy (ILAE) and the American Epilepsy Society (AES), in partnership with the National Institute of Neurological Disorders and Stroke (NINDS) and the National Institutes of Health (NIH) to define CDEs for animal epilepsy research studies and prepare guidelines for data collection and experimental procedures. If adopted, the preclinical CDEs could facilitate collaborative epilepsy research, comparisons of data across different laboratories, and promote rigor, transparency, and impact, particularly in therapy development.

The calcium-binding protein calbindin-D28k is critical for hippocampal function and cognition, but its expression is markedly decreased in various neurological disorders associated with epileptiform activity and seizures. In Alzheimer's disease (AD) and epilepsy, both of which are accompanied by recurrent seizures, the severity of cognitive deficits reflects the degree of calbindin reduction in the hippocampal dentate gyrus (DG). However, despite the importance of calbindin in both neuronal physiology and pathology, the regulatory mechanisms that control its expression in the hippocampus are poorly understood. Here we report an epigenetic mechanism through which seizures chronically suppress hippocampal calbindin expression and impair cognition. We demonstrate that DeltaFosB, a highly stable transcription factor, is induced in the hippocampus in mouse models of AD and seizures, in which it binds and triggers histone deacetylation at the promoter of the calbindin gene (Calb1) and downregulates Calb1 transcription. Notably, increasing DG calbindin levels, either by direct virus-mediated expression or inhibition of DeltaFosB signaling, improves spatial memory in a mouse model of AD. Moreover, levels of DeltaFosB and calbindin expression are inversely related in the DG of individuals with temporal lobe epilepsy (TLE) or AD and correlate with performance on the Mini-Mental State Examination (MMSE). We propose that chronic suppression of calbindin by DeltaFosB is one mechanism through which intermittent seizures drive persistent cognitive deficits in conditions accompanied by recurrent seizures.

The dentate gyrus (DG) principal cells are glutamatergic granule cells (GCs), and they are located in a compact cell layer. However, GCs are also present in the adjacent hilar region, but have been described in only a few studies. Therefore, we used the transcription factor prospero homeobox 1 (Prox1) to quantify GCs at postnatal day (PND) 16, 30, and 60 in a common mouse strain, C57BL/6J mice. At PND16, there was a large population of Prox1-immunoreactive (ir) hilar cells, with more in the septal than temporal hippocampus. At PND30 and 60, the size of the hilar Prox1-ir cell population was reduced. Similar numbers of hilar Prox1-expressing cells were observed in PND30 and 60 Swiss Webster mice. Prox1 is usually considered to be a marker of postmitotic GCs. However, many Prox1-ir hilar cells, especially at PND16, were not double-labeled with NeuN, a marker typically found in mature neurons. Most hilar Prox1-positive cells at PND16 co-expressed doublecortin (DCX) and calretinin, markers of immature GCs. Double-labeling with a marker of actively dividing cells, Ki67, was not detected. These results suggest that, surprisingly, a large population of cells in the hilus at PND16 are immature GCs (Type 2b and Type 3 cells). We also asked whether hilar Prox1-ir cell numbers are modifiable. To examine this issue, we conditionally deleted the proapoptotic gene BAX in Nestin-expressing cells at a time when there are numerous immature GCs in the hilus, PND2-8. When these mice were examined at PND60, the numbers of Prox1-ir hilar cells were significantly increased compared to control mice. However, deletion of BAX did not appear to change the proportion that co-expressed NeuN, suggesting that the size of the hilar Prox1-expressing population is modifiable. However, deleting BAX, a major developmental disruption, does not appear to change the proportion that ultimately becomes neurons.