Edward D. Plowey

Mutant LRRK2 enhances glutamatergic synapse activity and evokes excitotoxic dendrite degeneration

Mutations in leucine-rich repeat kinase 2 (LRRK2), which are associated with autosomal dominant Parkinsons disease, elicit progressive dendrite degeneration in neurons. We hypothesized that synaptic dysregulation contributes to mutant LRRK2-induced dendritic injury. We performed in vitro whole-cell voltage clamp studies of glutamatergic receptor agonist responses and glutamatergic synaptic activity in cultured rat cortical neurons expressing full-length wild-type and mutant forms of LRRK2. Expression of the pathogenic G2019S or R1441C LRRK2 mutants resulted in larger whole-cell current responses to direct application of AMPA and NMDA receptor agonists. In addition, mutant LRRK2-expressing neurons exhibited an increased frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in conjunction with increased excitatory synapse density as assessed by immunofluorescence for PSD95 and VGLUT1. Mutant LRRK2-expressing neurons showed enhanced vulnerability to acute synaptic glutamate stress. Furthermore, treatment with the NMDA receptor antagonist memantine significantly protected against subsequent losses in dendrite length and branching complexity. These data demonstrate an early association between mutant LRRK2 and increased excitatory synapse activity, implicating an excitotoxic contribution to mutant LRRK2 induced dendrite degeneration.

BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation

ABSTRACT The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267–337). Deletion of a BECN1 ECD subregion (amino acids 285–299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1s effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.

A Combination of Ontogeny and CNS Environment Establishes Microglial Identity

Microglia, the brains resident macrophages, are dynamic CNS custodians with surprising origins in the extra-embryonic yolk sac. The consequences of their distinct ontogeny are unknown but critical to understanding and treating brain diseases. We created a brain macrophage transplantation system to disentangle how environment and ontogeny specify microglial identity. We find that donor cells extensively engraft in the CNS of microglia-deficient mice, and even after exposure to a cell culture environment, microglia fully regain their identity when returned to the CNS. Though transplanted macrophages from multiple tissues can express microglial genes in the brain, only those of yolk-sac origin fully attain microglial identity. Transplanted macrophages of inappropriate origin, including primary human cells in a humanized host, express disease-associated genes and specific ontogeny markers. Through brain macrophage transplantation, we discover new principles of microglial identity that have broad applications to the study of disease and development of myeloid cell therapies.

Co-occurrence of a cerebral cavernous malformation and an orbital cavernous hemangioma in a patient with seizures and visual symptoms: Rare crossroads for vascular malformations.

Background: Cerebral cavernous malformations (CCMs) are angiographically occult vascular malformations of the central nervous system. As a result of hemorrhage and mass effect, patients may present with focal neurologic deficits, seizures, and other symptoms necessitating treatment. Once symptomatic, most often from hemorrhage, CCMs are treated with microsurgical resection. Orbital cavernous hemangiomas (OCHs) are similar but distinct vascular malformations that present within the orbital cavity. Even though CCMs and OCHs are both marked by dilated endothelial-lined vascular channels, they are infrequently seen in the same patient. Case Description: We provide a brief overview of the two related pathologies in the context of a patient presenting to our care with concomitant lesions, which were both resected in full without complication. Conclusion: This is the first known report that describes a case of concomitant CCM and OCH and explores the origins of two pathologies that are rarely encountered together in neurosurgical practice. Recognition of disparate symptomatologies is important for properly managing these patients.

GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes

Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12–ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

Hippocampal phospho-tau/MAPT neuropathology in the fornix in Alzheimer disease: an immunohistochemical autopsy study

Whereas early Alzheimer disease (AD) neuropathology and mild cognitive impairment are relatively common in aging, accurate prediction of patients that will progress to dementia requires new biomarkers. Recently, substantial work has focused on phospho-tau/MAPT (p-MAPT) neuropathology since its regional propagation correlates with the degree of cognitive impairment in AD. Recent diffusion tensor imaging studies in AD suggest that increased diffusion in the fornix secondary to p-MAPT-related axonal injury could serve as a predictive biomarker of the risk of disease progression. However, our knowledge of p-MAPT neuropathology in the fornix is limited. To address this gap in knowledge, we examined p-MAPT neuropathology in the fornix and basal forebrain nuclei via AT8 immunohistochemistry in 39 brain autopsies spanning the spectrum of AD neuropathologic changes. We found that the fornix and its precommissural efferent target nuclei (septum and nucleus accumbens) demonstrated neuronal and thread-like p-MAPT neuropathology only in National Institute on Aging/Alzheimer Association (NIA/AA) stages B2 and B3 of neurofibrillary degeneration, consistent with involvement after (and propagation from) the hippocampal formation. Interestingly, although tau astrogliopathy was frequently observed in the mammillary bodies in stage B2, neuronal tauopathy was not observed in the postcommissural targets (mammillary bodies and anterior thalamic nucleus) until stage B3. Tauopathy in the nucleus basalis of Meynert was strongly correlated with p-MAPT-positive axons in the fornix, suggesting that projections to the hippocampus also likely contribute to fornix tauopathy. Our cross-sectional autopsy findings indicate that the fornix is involved by p-MAPT neuropathology secondary to hippocampal involvement by AD neuropathology. Furthermore, our findings are compatible with the goal of in vivo detection of p-MAPT-related axonal pathology in the fornix in AD as a possible biomarker of p-MAPT progression from the hippocampal formation and underscore a need for additional clinical-radiologic-pathologic correlation studies.

The Impact of Latino Values and Cultural Beliefs on Brain Donation: Results of a Pilot Study to Develop Culturally Appropriate Materials and Methods to Increase Rates of Brain Donation in this Under-Studied Patient Group

ABSTRACT Objectives: Increasing the number of Latino persons with dementia who consent to brain donation (BD) upon death is an important public health goal that has not yet been realized. This study identified the need for culturally sensitive materials to answer questions and support the decision-making process for the family. Methods: Information about existing rates of BD was obtained from the Alzheimer’s Disease Centers. Several methods of data collection (query NACC database, contacting Centers, focus groups, online survey, assessing current protocol and materials) were used to give the needed background to create culturally appropriate BD materials. Results: A decision was made that a brochure for undecided enrollees would be beneficial to discuss BD with family members. For those needing further details, a step-by-step handout would provide additional information. Conclusions: Through team collaboration and engagement of others in the community who work with Latinos with dementia, we believe this process allowed us to successfully create culturally appropriate informational materials that address a sensitive topic for Hispanic/Latino families. Clinical Implications: Brain tissue is needed to further knowledge about underlying biological mechanism of neurodegenerative diseases, however it is a sensitive topic. Materials assist with family discussion and facilitate the family’s follow-through with BD.

RARE MISSENSE VARIANTS ON ZNF679 AND CTD-3214H19.16 SEGREGATE IN A FAMILY WITH A HISTORY OF SYNUCLEINOPATHY

profiling indicated that running significantly increases transcripts related to angiogenesis and vascular remodeling. To evaluate exercise as a therapeutic, we will provide a running wheel to determine whether running can preserve healthy vasculature in mice carrying genetic risk factors for dementia. Results:Preliminary data suggests increased vascular dysfunction two month old as characterized by extravascular fibrin in B6.Apoe4/4 compared to B6 controls. RNA-seq analysis of mice run at a young age resulted in Gene Ontology terms associated with angiogenesis and vascular remodeling (ie. Mmp14, Angpt1, Angpt2, Vegfa). Additional data will also be presented regarding embryonic vascular development, and the effects of running on angiogenesis and vascular remodeling. Conclusions:This work is providing novel evaluation of the earliest effects of Apoe on the cerebrovasculature throughout development as well as assessing running as an intervention to strengthen vascular integrity for those with a genetic predisposition for dementia.

FEASIBILITY OF USING X-RAY FLUORESCENCE IMAGING AND ABSORPTION SPECTROSCOPY TO EVALUATE IRON DISTRIBUTION AND OXIDATIVE STATE IN THE ALZHEIMER'S DISEASE HIPPOCAMPUS

(NanoSIMS) in a combined approach we term SILK-SIMS. Results: 1) In human AD brain, the incorporation of tracer into plaques in a participant with clinical stage AD dementia suggests that plaques can be dynamic with activity over days even in later stages of disease. 2) Tracer enrichment was highly variable and asymmetric with both quiescent and active nanometer sub-regions of tracer incorporation. 3) Neurons are hyper-metabolic cells compared to surrounding neuropil. We will demonstrate whether disturbed neuronal metabolism (a proxy for function) may be indicative of declining neuronal health and whether this is correlated with the presence of tau aggregates. Conclusions: SILK-SIMS studies will provide invaluable information on plaque dynamics and neuronal health, and tau aggregation in the normal and diseased brain. These studies will offer new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.

CORRELATIVE MRI/OPTICAL/ELECTRON MICROSCOPY EVALUATION OF METAL DISTRIBUTION AND OXIDATIVE STATE IN THE ALZHEIMER’S HIPPOCAMPUS

USA; William S. Middleton Memorial Veterans Hospital, Madison, WI, USA; University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; University of Wisconsin,Madison,Madison, WI, USA; Wisconsin Alzheimer’s Disease Research Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; University of California-Irvine, Irvine, CA, USA; Geriatric Research Education and Clinical Center, Wm. S. Middleton Veterans Hospital, Madison, WI, USA. Contact e-mail: ksprecher@wisc.edu