Gwenn A. Garden

There is an urgent need to improve dementia ascertainment robustness in real-world studies assessing drug effects on dementia risk. We developed algorithms to dementia identification algorithms using Medicare claims (inpatient/outpatient/prescription) from 3318 Visit 5 (2011-2013) and 1828 Visit 6 (2016-2017) participants of the Atherosclerosis Risk in Communities (ARIC) Study, validated against ARIC's rigorous syndromic dementia classification. Algorithm performance was compared to existing algorithms (Jain, Bynum, Lee). We further evaluated algorithms effectiveness in a 20% random Medicare sample aged ≥70 years who initiating liraglutide or dipeptidyl peptidase 4 inhibitors (DPP4i) to assess 3-year adjusted risk difference (aRD) for dementia. Our incident dementia algorithm required two dementia diagnostic codes within 1-year, or one dementia code plus a new dementia prescription within 90 days. It achieved a positive predictive value (PPV) of 69.2%, specificity of 99.0%, and sensitivity of 34.6% (population prevalence: 8.8%), comparable to extant algorithms (PPV, 58.7–68.6%; sensitivity 25.5–40.4%). Prevalent dementia algorithm (without requiring incident diagnoses/prescriptions) demonstrated similar performance. In the Medicare sample, dementia risk ranged from 3.0% to 12.5%, aRD comparing liraglutide to DPP4i varied −1.2% to −3.6%, with our algorithm closely matching the Bynum algorithm. Algorithm selection significantly impacts treatment effect estimates, highlighting its importance in in pharmacoepidemiologic research.

Alzheimer’s Disease (AD) is characterized by the accumulation of extracellular amyloid-β (Aβ) as well as CNS and systemic inflammation. Microglia, the myeloid cells resident in the CNS, use microRNAs to rapidly respond to inflammatory signals. MicroRNAs (miRNAs) modulate inflammatory responses in microglia, and miRNA profiles are altered in Alzheimer’s disease (AD) patients. Expression of the pro-inflammatory miRNA, miR-155, is increased in the AD brain. However, the role of miR-155 in AD pathogenesis is not well-understood. We hypothesized that miR-155 participates in AD pathophysiology by regulating microglia internalization and degradation of Aβ. We used CX3CR1^CreER/+ to drive-inducible, microglia-specific deletion of floxed miR-155 alleles in two AD mouse models. Microglia-specific inducible deletion of miR-155 in microglia increased anti-inflammatory gene expression while reducing insoluble Aβ_1-42 and plaque area. Yet, microglia-specific miR-155 deletion led to early-onset hyperexcitability, recurring spontaneous seizures, and seizure-related mortality. The mechanism behind hyperexcitability involved microglia-mediated synaptic pruning as miR-155 deletion altered microglia internalization of synaptic material. These data identify miR-155 as a novel modulator of microglia Aβ internalization and synaptic pruning, influencing synaptic homeostasis in the setting of AD pathology.

Background: Computational phenotypes are most often combinations of patient billing codes that are highly predictive of disease using electronic health records (EHR). In the case of rare diseases that can only be diagnosed by genetic testing, computational phenotypes identify patient cohorts for genetic testing and possible diagnosis. This article details the validation of a computational phenotype for PTEN hamartoma tumor syndrome (PHTS) against the EHR of patients at three collaborating clinical research centers: Boston Children's Hospital, Children's National Hospital, and the University of Washington. Methods: A combination of billing codes from the International Classification of Diseases versions 9 and 10 (ICD-9 and ICD-10) for diagnostic criteria postulated by a research team at Cleveland Clinic was used to identify patient cohorts for genetic testing from the clinical data warehouses at the three research centers. Subsequently, the EHR—including billing codes, clinical notes, and genetic reports—of these patients were reviewed by clinical experts to identify patients with PHTS. Results: The PTEN genetic testing yield of the computational phenotype, the number of patients who needed to be genetically tested for incidence of pathogenic PTEN gene variants, ranged from 82 to 94% at the three centers. Conclusions: Computational phenotypes have the potential to enable the timely and accurate diagnosis of rare genetic diseases such as PHTS by identifying patient cohorts for genetic sequencing and testing.

Microglia maintain brain health and play important roles in disease and injury. Despite their known ability to proliferate, the precise nature of the population(s) capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (CD133 or Prom1) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1 expressing cells isolated from mixed cortical cultures will generate new microglia in vitro. To determine whether Prom1 expressing cells generate new microglia in vivo, we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1 expressing cells lead to the expression of TdTomato in all Prom1 expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato expressing microglia at six months of age that increased in size at nine months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1 expressing progenitors was observed with the majority of new microglia derived from Prom1 negative cells. Together, these findings reveal that Prom1 expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo. Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1 expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain, but not in response to ischemia/reperfusion. SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the central nervous system, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia.

Immunotherapy for hematologic malignancies with CD19-directed CAR T cells is associated with neurotoxicity in about 40% of patients. Systemic cytokine release syndrome, endothelial activation, and disruption of endothelial integrity have all been associated with neurotoxicity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction. We developed a syngeneic mouse model which manifests systemic cytokine release and behavioral abnormalities within 3-5 days after infusion of high-dose murine CD19-CAR T cells. Histologic examination revealed widespread brain hemorrhages, diffuse extravascular IgG deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries. In vivo two-photon imaging of blood flow revealed plugging of >10% of capillaries by leukocytes, associated with regions of localized hypoxia. These data reveal capillary obstruction and associated brain hypoxia and microvascular decline as a potential basis for neurotoxicity during CD19-CAR T cell treatment in humans, which may be amenable to therapeutic interventions.

Microglia are the innate immune cells of the central nervous system that adopt rapid functional changes in response to Damage Associated Molecular Patterns, including aggregated β‐Amyloid (Aβ) found in Alzheimer's disease (AD). microRNAs (miRNAs) are post‐transcriptional modulators that influence the timing and magnitude of microglia inflammatory responses by downregulating the expression of inflammatory effectors. Recent studies implicate miR‐155, a miRNA known to regulate inflammatory responses, in the pathogenesis of neurodegenerative disorders including multiple sclerosis, ALS, familial Parkinson's disease, and AD. In this work, we asked if miR‐155 expression in microglia modifies cellular behaviors in response to fibrillar Aβ_1‐42 (fAβ_1‐42), in vitro. We hypothesized that in microglia, miR‐155 expression would impact the internalization and catabolism of extracellular fAβ_1‐42. Primary microglia stimulated with lipopolysaccharide demonstrate fast upregulation of miR‐155 followed by delayed upregulation of miR‐146a, an anti‐inflammatory miRNA. Conditional overexpression of miR‐155 in microglia resulted in significant upregulation of miR‐146a. Conditional deletion of miR‐155 promoted transit of fAβ_1‐42 to low‐pH compartments where catabolism occurs, while miR‐155 overexpression decreases fAβ_1‐42 catabolism. Uptake of fAβ_1‐42 across the plasma membrane increased with both up and downregulation of miR‐155 expression. Taken together, our results support the hypothesis that inflammatory signaling influences the ability of microglia to catabolize fAβ_1‐42 through interconnected mechanisms modulated by miR‐155. Understanding how miRNAs modulate the ability of microglia to catabolize fAβ_1‐42 will further elucidate the role of cellular players and molecular crosstalk in AD pathophysiology.

Repeated cycles of alcohol intoxication and withdrawal induce profound changes in gene expression that can contribute to the physiological and behavioral consequences of ethanol. Since neuroinflammation is an important consequence of these changes, we used a novel strategy to investigate the impact of repeated cycles of chronic intermittent ethanol vapor and withdrawal on the RNAs actively undergoing translation in microglia in striatum, a key region involved in the relapse to ethanol consumption. We performed deep sequencing of the “translatome” from striatal microglia of male and female RiboTag mice, yielding a snapshot of RNA translation during alcohol intoxication and after 8 hours of withdrawal. Chronic intermittent ethanol produced robust changes in the translatome, with increases in genes and pathways associated with cytokine signaling, indicating increased neuroinflammation and microglial activation. After 8 hours of ethanol withdrawal, many inflammatory pathways remained upregulated and phagocytotic and proapoptotic pathways were increased. Using unbiased network analysis, we identified gene modules that were differentially expressed in ethanol intoxicated vs. withdrawing animals. Genes associated with the unfolded protein response (UPR) were over-represented in one such module after withdrawal, including the transcription factor Ddit3 (CHOP), an important mediator of the UPR. We tested the impact of conditional knockout of CHOP from microglia specifically; following withdrawal from chronic intermittent ethanol, these mice had reduced thermoregulatory disturbances, anxiety-like behavior, and voluntary ethanol consumption compared to wild-type littermates. We conclude that CHOP and the UPR in microglia may be important targets for reducing the impact of withdrawal from chronic ethanol exposure.

Histone deacetylases (HDACs) catalyze acetyl group removal from histone proteins, leading to altered chromatin structure and gene expression. HDAC2 is highly expressed in adult brain, and HDAC2 levels are elevated in Alzheimer's disease (AD) brain. We previously reported that neuron‐specific splice isoforms of Endophilin‐B1 (Endo‐B1) promote neuronal survival, but are reduced in human AD brain and mouse models of AD and stroke. Here, we demonstrate that HDAC2 suppresses Endo‐B1 expression. HDAC2 knockdown or knockout enhances expression of Endo‐B1. Conversely, HDAC2 overexpression decreases Endo‐B1 expression. We also demonstrate that neurons exposed to beta‐amyloid increase HDAC2 and reduce histone H3 acetylation while HDAC2 knockdown prevents Aβ induced loss of histone H3 acetylation, mitochondrial dysfunction, caspase‐3 activation, and neuronal death. The protective effect of HDAC2 knockdown was abrogated by Endo‐B1 shRNA and in Endo‐B1‐null neurons, suggesting that HDAC2‐induced neurotoxicity is mediated through suppression of Endo‐B1. HDAC2 overexpression also modulates neuronal expression of mitofusin2 (Mfn2) and mitochondrial fission factor (MFF), recapitulating the pattern of change observed in AD. HDAC2 knockout mice demonstrate reduced injury in the middle cerebral artery occlusion with reperfusion (MCAO/R) model of cerebral ischemia demonstrating enhanced neuronal survival, minimized loss of Endo‐B1, and normalized expression of Mfn2. These findings support the hypothesis that HDAC2 represses Endo‐B1, sensitizing neurons to mitochondrial dysfunction and cell death in stroke and AD.