Faculty Bibliography

Neurofilament and tau proteins are neuron-specific cytoskeletal proteins that are enriched in axons, regulated by many of the same protein kinases, interact physically, and are the principal constituents of neurofibrillary lesions in major adult-onset dementias. Both proteins share functions related to the modulation of stability and functions of the microtubule network in axons, axonal transport and scaffolding of organelles, long-term synaptic potentiation, and learning and memory. Expression of these proteins is regulated not only at the transcriptional level but also through posttranscriptional control of pre-mRNA splicing, mRNA stability, transport, localization, local translation and degradation. Current evidence suggests that posttranscriptional determinants of their levels are usually regulated by RNA-binding proteins and microRNAs primarily through 3'-untranslated regions of neurofilament and tau mRNAs. Dysregulations of neurofilament and tau expression caused by mutations or pathologies of RNA-binding proteins such as TDP43, FUS and microRNAs are increasingly recognized in association with varied neurological disorders. In this review, we summarize the current understanding of posttranscriptional control of neurofilament and tau by examining the posttranscriptional regulation of neurofilament and tau by RNA-binding proteins and microRNAs implicated in health and diseases.

Human apolipoprotein E (APOE) is the greatest determinant of genetic risk for memory deficits and Alzheimer's disease (AD). While APOE4 drives memory loss and high AD risk, APOE2 leads to healthy brain aging and reduced AD risk compared to the common APOE3 variant. We examined brain APOE protein levels in humanized mice homozygous for these alleles and found baseline levels to be age- and isoform-dependent: APOE2 levels were greater than APOE3, which were greater than APOE4. Despite the understanding that APOE lipoparticles do not traverse the blood-brain barrier, we show that brain APOE levels are responsive to dietary fat intake. Challenging mice for 6 months on a Western diet high in fat and cholesterol increased APOE protein levels in an allele-dependent fashion with a much greater increase within blood plasma than within the brain. In the brain, APOE2 levels responded most to the Western diet challenge, increasing by 20 % to 30 %. While increased lipoparticles are generally deleterious in the periphery, we propose that higher brain APOE2 levels may represent a readily available pool of beneficial lipid particles for neurons.

Linear regression is one of the most used statistical techniques in neuroscience, including the study of the neuropathology of Alzheimer's disease (AD) dementia. However, the practical utility of this approach is often limited because dependent variables are often highly skewed and fail to meet the assumption of normality. Applying linear regression analyses to highly skewed datasets can generate imprecise results, which lead to erroneous estimates derived from statistical models. Furthermore, the presence of outliers can introduce unwanted bias, which affect estimates derived from linear regression models. Although a variety of data transformations can be utilized to mitigate these problems, these approaches are also associated with various caveats. By contrast, a robust regression approach does not impose distributional assumptions on data allowing for results to be interpreted in a similar manner to that derived using a linear regression analysis. Here, we demonstrate the utility of applying robust regression to the analysis of data derived from studies of human brain neurodegeneration where the error distribution of a dependent variable does not meet the assumption of normality. We show that the application of a robust regression approach to two independent published human clinical neuropathologic data sets provides reliable estimates of associations. We also demonstrate that results from a linear regression analysis can be biased if the dependent variable is significantly skewed, further indicating robust regression as a suitable alternate approach.

Background: Alzheimer"™s disease (AD) is a neurodegenerative illness characterized by progressive accumulation of amyloid beta (Aβ) and neurofibrillary tangles, with cognitive impairment and altered neural activity. Hyperexcitability in the early stages of AD contribute to Aβ accumulation and cognitive impairment, aggravating the progression of AD. However, the hyperexcitability origin is not clear. This study aimed to test whether mossy cells (MCs), an excitatory cell of the hippocampal dentate gyrus, show increased excitability at early stages of AD and contribute to the increased network excitability generation. Indeed, alterations of MCs contribute to hyperexcitability and cognitive impairment in epilepsy. However, the role of MCs in AD has not been substantially explored. Methods: Intrinsic and synaptic properties of MCs and granule cells (GCs) from WT and Tg2576 mice at early ages (1-2 m.o.) were characterized by whole-cell patch-clamp recordings. Synaptic properties included the frequency and amplitude of spontaneous excitatory postsynaptic potentials (EPSPs) and excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs). Deterioration in MCs morphology was evaluated using Nissl staining and GluR2/3 labeling by light- and confocal microscopy. Aβ deposition was evaluated using the McSA1 antibody. Results: Tg2576 GCs did not have any significant difference in their intrinsic properties, as we shown previously in mice ∼3 m.o. However, an enhanced excitatory and inhibitory input to GCs, depicted by augmented IPSC (7.16 vs 14.04 events/s) and NMDA-mediated EPSC frequencies (0.81 vs 1.41 events/s) were found. Interestingly, Tg2576 MCs had an augmented EPSP frequency (5.75 vs 9.44 events/s), and their intrinsic properties showed a depolarized RMP (-72.88 vs -58.36 mV), and reduced rheobase (145.56 vs 47.14 pA), AP amplitude (98.14 vs 76.66 mV), time-to-peak (552.75 vs 266.16 ms) and maximum rise (171.44 vs 88.68 mV/ms) and decay slopes (-61.17 vs -42.38 mV/ms). The correlation between #APs and current injected showed Tg2576 MCs fired significantly more APs (SEZD = 0.34; z = 2.48). Tg2576 MCs showed robust intracellular Aβ aggregation without any significant morphological change. Conclusions: MCs changes in excitability and early accumulation of Aβ suggest that MCs could be the cause of increased excitability occurring later in GCs. In this manner, MCs could be an important contributor to AD.

Mechanisms of Alzheimer's disease (AD) and its putative prodromal stage, amnestic mild cognitive impairment (aMCI), involve the dysregulation of multiple candidate molecular pathways that drive selective cellular vulnerability in cognitive brain regions. However, the spatiotemporal overlap of markers for pathway dysregulation in different brain regions and cell types presents a challenge for pinpointing causal versus epiphenomenal changes characterizing disease progression. To approach this problem, we performed Weighted Gene Co-expression Network Analysis and STRING interactome analysis of gene expression patterns quantified in frontal cortex samples (Brodmann area 10) from subjects who died with a clinical diagnosis of no cognitive impairment, aMCI, or mild/moderate AD. Frontal cortex was chosen due to the relatively protracted involvement of this region in AD, which might reveal pathways associated with disease onset. A co-expressed network correlating with clinical diagnosis was functionally associated with insulin signaling, with insulin (INS) being the most highly connected gene within the network. Co-expressed networks correlating with neuropathological diagnostic criteria (e.g., NIA-Reagan Likelihood of AD) were associated with platelet-endothelium-leucocyte cell adhesion pathways and hypoxia-oxidative stress. Dysregulation of these functional pathways may represent incipient alterations impacting disease progression and the clinical presentation of aMCI and AD.

Extracellular vesicles (EVs) are nanoscale vesicles secreted into the extracellular space by all cell types, including neurons and astrocytes in the brain. EVs play pivotal roles in physiological and pathophysiological processes such as waste removal, cell-to-cell communication and transport of either protective or pathogenic material into the extracellular space. Here we describe a detailed protocol for the reliable and consistent isolation of EVs from both murine and human brains, intended for anyone with basic laboratory experience and performed in a total time of 27 h. The method includes a mild extracellular matrix digestion of the brain tissue, a series of filtration and centrifugation steps to purify EVs and an iodixanol-based high-resolution density step gradient that fractionates different EV populations, including mitovesicles, a newly identified type of EV of mitochondrial origin. We also report detailed downstream protocols for the characterization and analysis of brain EV preparations using nanotrack analysis, electron microscopy and western blotting, as well as for measuring mitovesicular ATP kinetics. Furthermore, we compared this novel iodixanol-based high-resolution density step gradient to the previously described sucrose-based gradient. Although the yield of total EVs recovered was similar, the iodixanol-based gradient better separated distinct EV species as compared with the sucrose-based gradient, including subpopulations of microvesicles, exosomes and mitovesicles. This technique allows quantitative, highly reproducible analyses of brain EV subtypes under normal physiological processes and pathological brain conditions, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

Extracellular vesicles (EVs) in the brain play a role in neuronal homeostasis by removing intracellular material and regulating cell-to-cell communication. Given that sex and aging differentially modulate brain networks, we investigated sex-dependent differences in EV levels and content in the brain during aging. EVs were isolated from the brains of 3, 6, 12, 18, and 24 month-old female and male C57BL/6 J mice, and the levels of different EV species determined. While the number of plasma membrane-derived microvesicles and a subset of late endosomes-derived exosomes increased with age in the brain of female mice, no significant changes were seen in males. Mitochondria-derived mitovesicles in the brain increased during aging in both sexes, a change that may reflect aging-dependent alterations in mitochondrial function. These findings reveal enhanced turnover during aging in female brains, suggesting a mechanism for advantageous successful female brain aging and sex-depending different susceptibility to age-related neurodegenerative diseases.

The hippocampal CA2 region, an area important for social memory, has been suspected to play a role in temporal lobe epilepsy (TLE) because of its resistance to degeneration observed in neighboring CA1 and CA3 regions in both humans and rodent models of TLE. However, little is known about whether alterations in CA2 properties promote seizure generation or propagation. Here, we addressed the role of CA2 using the pilocarpine-induced status epilepticus model of TLE. Ex vivo electrophysiological recordings from acute hippocampal slices revealed a set of coordinated changes that enhance CA2 PC intrinsic excitability, reduce CA2 inhibitory input, and increase CA2 excitatory output to its major CA1 synaptic target. Moreover, selective chemogenetic silencing of CA2 pyramidal cells caused a significant decrease in the frequency of spontaneous seizures measured in vivo. These findings provide the first evidence that CA2 actively contributes to TLE seizure activity and may thus be a promising therapeutic target.