The serotonin (5-hydroxytryptamine, 5-HT) transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) is thought to alter 5-HT signaling and contribute to behavioral and cognitive phenotypes in depression as well as Alzheimer disease (AD). We explored how well the short (S) and long (L) alleles of the 5-HTTLPR align with serotoninergic indices in 60 autopsied cortical samples from early-onset AD/EOAD and late-onset AD/LOAD donors, and age- and sex-matched controls. Stratifying data by either diagnosis-by-genotype or by sex-by-genotype revealed that the donor's 5-HTTLPR genotype, i.e., L/L, S/L, or S/S, did not affect 5-HTT mRNA or protein expression. However, the glycosylation of 5-HTT was significantly higher in control female (vs. male) samples and tended to decrease in female EOAD/LOAD samples, but remained unaltered in male LOAD samples. Glycosylated forms of the vesicular monoamine transporter (VMAT2) were lower in both male and female AD samples, while a sex-by-genotype stratification revealed a loss of VMAT2 glycosylation specifically in females with an L/L genotype. VMAT2 and 5-HTT glycosylation were correlated in male samples and inversely correlated in female samples in both stratification models. The S/S genotype aligned with lower levels of 5-HT turnover in females (but not males) and with an increased glycosylation of the post-synaptic 5-HT2C receptor. Interestingly, the changes in presynaptic glycosylation were evident primarily in female carriers of the APOE ε4 risk factor for AD. Our data do not support an association between 5-HTTLPR genotype and 5-HTT expression, but they do reveal a non-canonical association of 5-HTTLPR genotype with sex-dependent glycosylation changes in pre- and post-synaptic markers of serotoninergic neurons. These patterns of change suggest adaptive responses in 5-HT signaling and could certainly be contributing to the female prevalence in risk for either depression or AD.

Background and Objective: Image-guided neurosurgery uses information from a wide spectrum of methods to inform the neurosurgeon's judgement about which tissue to resect and which to spare. Imaging data are registered to the patient's skull so that they correspond to the intraoperative macro- and microscopic view. The correspondence between imaging and optical systems breaks down during surgery, however, as a result of cerebro-spinal fluid drain age, tissue resection, and gravity-based brain shift. In this work we investigate whether a map of surface veins, automatically segmented from MRI, could serve as additional reference system. Methods: Gradient-echo based [Formula: see text]-weighted imaging was performed on two human cadavers heads using a 7 Tesla MRI scanner. Automatic vessel segmentation was performed using the Frangi vesselness filter, and surface renderings of vessels compared with photographs of the surface of the brain following craniotomy. Results: A high level of correspondence was established between vessel maps and the post autopsy photographs. Corresponding veins, including the prominent superior anastomotic veins, could be identified in all brain lobes. Conclusion: Automatic surface vessel segmentation is feasible and the high correspondence to post autopsy photographs indicates that they could be used as an additional reference system for image-guided neurosurgery in order to maintain the correspondence between imaging and optical systems.This has the advantage over a skull-based reference system that veins are clearly visible to the surgeon and move and deform with the underlying tissue, potentially making this surface net of landmarks robust to brain shift.

Chronic inflammation is a common phenomenon present in the background of multiple neurodegenerative diseases, including Alzheimer's disease (AD). The arachidonic acid pathway overproduces proinflammatory eicosanoids during these states and glial cells in the brain gradually lose their vital functions of protecting and supporting neurons. In this study, the role of different key enzymes of the eicosanoid pathway mediating inflammatory responses was examined in vitro and ex vivo using human fetal glial cells. Astrocytes and microglia were exposed to proinflammatory agents i.e., cytokines interleukin 1-β (IL-1β) and tumor necrosis factor (TNF-α). ELISA assays were used to examine the effects of inhibitors of key enzymes in the eicosanoid pathway. Inhibitors for 5-lipoxygenase (5-LOX) and cyclo-oxygenase 2 (COX-2) in both cell types and 5-, 12-, and 15-LOX-inhibitor in astrocytes reduced significantly IL-6 secretion, compared to exposed glial cells without inhibitors. The cytokine antibody array showed that especially treatments with 5, -12, and -15 LOX inhibitor in astrocytes, 5-LOX inhibitor in microglia and COX-2 inhibitor in both glial cell types significantly reduced the expression of multiple proinflammatory cytokines. Furthermore, human fetal astrocytes and microglia were cultured on top of AD-affected and control human brain sections for 30 h. According to the immunochemical evaluation of the level of total Aβ, astrocytes were very efficient at degrading Aβ from AD-affected brain sections ex vivo; simultaneously added enzyme inhibitors did not increase their Aβ degradation capabilities. Microglia were not able to reduce the level of total Aβ during the 30 h incubation time.

TAR DNA-binding protein 43 (TDP-43) is the major component of the ubiquitin-positive protein aggregates seen in the majority of frontotemporal lobar degeneration and amyotrophic lateral sclerosis cases. TDP-43 belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is involved in the regulation of RNA transcription, splicing, transport and translation. There are a great many hnRNPs, which often have overlapping functions and act cooperatively in RNA processing. Here we demonstrate that another hnRNP family member, hnRNP E2, shows a striking accumulation within dystrophic neurites and cytoplasmic inclusions in the frontal cortex and hippocampus of a subset of FTLD-TDP cases belonging to pathological subtypes A and C, where hnRNP E2 was found to co-localize with 87% of TDP-43 immunopositive inclusions. hnRNP E2-positive inclusions were not seen in FTLD-TDP cases with the C9orf72 expansion or in any other neurodegenerative disorders examined. This interaction with TDP-43 in specific FTLD subtypes suggests different underlying neurodegenerative pathways.

Abnormal endo-lysosomal morphology is an early cytopathological feature of Alzheimer's disease (AD) and genome-wide association studies (GWAS) have implicated genes involved in the endo-lysosomal network (ELN) as conferring increased risk for developing sporadic, late-onset AD (LOAD). Characterization of ELN pathology and the underlying pathophysiology is a promising area of translational AD research and drug development. However, rigorous study of ELN vesicles in AD and aged control brains poses a unique constellation of methodological challenges due in part to the small size of these structures and subsequent requirements for high-resolution imaging. Here we provide a detailed protocol for high-resolution 3D morphological quantification of neuronal endosomes in postmortem AD brain tissue, using immunofluorescent staining, confocal imaging with image deconvolution, and Imaris software analysis pipelines. To demonstrate these methods, we present neuronal endosome morphology data from 23 sporadic LOAD donors and one aged non-AD control donor. The techniques described here were developed across a range of AD neuropathology to best optimize these methods for future studies with large cohorts. Application of these methods in research cohorts will help advance understanding of ELN dysfunction and cytopathology in sporadic AD.

In humans, a considerable number of the autopsy samples of cognitively normal individuals aged between 57 and 102 years have revealed the presence of amyloid plaques, one of the typical signs of AD, indicating that many of us use mechanisms that defend ourselves from the toxic consequences of Aß. The human APP NL/F (hAPP NL/F) knockin mouse appears as the ideal mouse model to identify these mechanisms, since they have high Aß42 levels at an early age and moderate signs of disease when old. Here we show that in these mice, the brain levels of the hemoprotein Neuroglobin (Ngb) increase with age, in parallel with the increase in Aß42. In vitro, in wild type neurons, exogenous Aß increases the expression of Ngb and Ngb over-expression prevents Aß toxicity. In vivo, in old hAPP NL/F mice, Ngb knockdown leads to dendritic tree simplification, an early sign of Alzheimer's disease. These results could indicate that Alzheimer's symptoms may start developing at the time when defense mechanisms start wearing out. In agreement, analysis of plasma Ngb levels in aged individuals revealed decreased levels in those whose cognitive abilities worsened during a 5-year longitudinal follow-up period.

Evidence of inflammation has been consistently associated with pathology in Parkinson's disease (PD)-affected brains, and has been suggested as a causative factor. Dopaminergic neurons in the substantia nigra (SN) pars compacta, whose loss results in the clinical symptoms associated with PD, are particularly susceptible to inflammatory damage and oxidative stress. Inflammation in the striatum, where SN dopaminergic neurons project, is also a feature of PD brains. It is not known whether inflammatory changes occur first in striatum or SN. Many animal models of PD have implicated certain inflammatory molecules with dopaminergic cell neuronal loss; however, there have been few studies to validate these findings by measuring the levels of these and other inflammatory factors in human PD brain samples. This study also included samples from incidental Lewy body disease (ILBD) cases, since ILBD is considered a non-symptomatic precursor to PD, with subjects having significant loss of tyrosine hydroxylase-producing neurons. We hypothesized that there may be a progressive change in key inflammatory factors in ILBD samples intermediate between neurologically normal and PD. To address this, we used a quantitative antibody-array platform (Raybiotech-Quantibody arrays) to measure the levels of 160 different inflammation-associated cytokines, chemokines, growth factors, and related molecules in extracts of SN and striatum from clinically and neuropathologically characterized PD, ILBD, and normal control cases. Patterns of changes in inflammation and related molecules were distinctly different between SN and striatum. Our results showed significantly different levels of interleukin (IL)-5, IL-15, monokine induced by gamma interferon, and IL-6 soluble receptor in SN between disease groups. A different panel of 13 proteins with significant changes in striatum, with IL-15 as the common feature, was identified. Although the ability to detect some proteins was limited by sensitivity, patterns of expression indicated involvement of certain T-cell cytokines, vascular changes, and loss of certain growth factors, with disease progression. The results demonstrate the feasibility of profiling inflammatory molecules using diseased human brain samples, and have provided additional targets to validate in relation to PD pathology.

This study examines the atrophy patterns in the entorhinal and transentorhinal cortices of subjects that converted from normal cognition to mild cognitive impairment. The regions were manually segmented from 3T MRI, then corrected for variability in boundary definition over time using an automated approach called longitudinal diffeomorphometry. Cortical thickness was calculated by deforming the gray matter-white matter boundary surface to the pial surface using an approach called normal geodesic flow. The surface was parcellated based on four atlases using large deformation diffeomorphic metric mapping. Average cortical thickness was calculated for (1) manually-defined entorhinal cortex, and (2) manually-defined transentorhinal cortex. Group-wise difference analysis was applied to determine where atrophy occurred, and change point analysis was applied to determine when atrophy started to occur. The results showed that by the time a diagnosis of mild cognitive impairment is made, the transentorhinal cortex and entorhinal cortex was up to 0.6 mm thinner than a control with normal cognition. A change point in atrophy rate was detected in the transentorhinal cortex 9-14 years prior to a diagnosis of mild cognitive impairment, and in the entorhinal cortex 8-11 years prior. The findings are consistent with autopsy findings that demonstrate neuronal changes in the transentorhinal cortex before the entorhinal cortex.

Electrical stimulation of peripheral nerves by implanted electrodes is an effective treatment for certain pelvic floor diseases. As well as intravesical electrical stimulation, this predominantly includes stimulation of the sacral nerve, tibial nerve, and pudendal nerve. The pudendal nerve is one of the main nerves that stimulate pelvic floor muscles, external urethral meatus, and the anal sphincter and pelvic organs, and it may have effects on frequent urination, urgency, dysuria, and perineal pain. It is difficult to locate because of its anatomical course, however, leading to difficulties fixing the electrode, which increases the difficulty of pudendal nerve electrical stimulation in clinical practice. In the current study 3D printed navigation was used to solve these problems. Combined with autopsy data and patient pelvic and nerve data, a personalized design was generated. Neural modulation of the pudendal nerve was achieved by implanting the lead with the guidance of 3D printed navigation. 3D printed navigation can maximize the phase II conversion rate, reduce the difficulty of surgery, shorten the operation time, reduce damage to additional organs and blood vessels, and increase the accuracy of electrode implantation, and it can be performed while the patient is awake. It is an accurate, reversible, efficient, and minimally invasive surgery.

Pathological hallmarks of amyotrophic lateral sclerosis (ALS), including protein misfolding, are well established in oligodendrocytes. More recently, an RNA trafficking deficit of key myelin proteins has been suggested in oligodendrocytes in ALS but the extent to which this affects myelination and the relative contribution of this to disease pathogenesis is unclear. ALS autopsy research findings showing demyelination contrasts with the routine clinical-pathological workup of ALS cases where it is rare to see white matter abnormalities other than simple Wallerian degeneration secondary to widespread neuronal loss. To begin to address this apparent variance, we undertook a comprehensive evaluation of myelination at an RNA, protein and structural level using human pathological material from sporadic ALS patients, genetic ALS patients (harboring C9orf72 mutation) and age- and sex-matched non-neurological controls. We performed (i) quantitative spatial profiling of the mRNA transcript encoding myelin basic protein (MBP), (ii) quantification of MBP protein and (iii) the first quantitative structural assessment of myelination in ALS post-mortem specimens by electron microscopy. We show no differences in MBP protein levels or ultrastructural myelination, despite a significant dysregulation in the subcellular trafficking of MBP mRNA in ALS patients compared to controls. We therefore confirm that whilst there are cell autonomous mRNA trafficking deficits affecting oligodendrocytes in ALS, this has no effect on myelin structure.

Parkinson's disease (PD) and dementia with Lewy body (DLB) are the most common synucleinopathies. SNCA gene is a major genetic risk factor for these diseases group, and dysregulation of its expression has been implicated in the genetic etiologies of several synucleinopathies. DNA methylation at CpG island (CGI) within SNCA intron 1 has been suggested as a regulatory mechanism of SNCA expression, and changes in methylation levels at this region were associated with PD and DLB. However, the role of DNA methylation in the regulation of SNCA expression in a cell-type specific manner and its contribution to the pathogenesis of PD and DLB remain poorly understood, and the data are conflicting. Here, we employed a bisulfite pyrosequencing technique to profile the DNA methylation across SNCA intron 1 CGI in PD and DLB compared to age- and sex-matched normal control subjects. We analyzed homogenates of bulk post-mortem frozen frontal cortex samples and a subset of neuronal and glia nuclei sorted by the fluorescence-activated nuclei sorting (FANS) method. Bulk brain tissues showed no significant difference in the overall DNA methylation across SNCA intron 1 CGI region between the neuropathological groups. Sorted neuronal nuclei from PD frontal cortex showed significant lower levels of DNA methylation at this region compared to normal controls, but no differences between DLB and control, while sorted glia nuclei exhibited trends of decreased overall DNA methylation in DLB only. In conclusion, our data suggested disease-dependent cell-type specific differential DNA methylation within SNCA intron 1 CGI. These changes may affect SNCA dysregulation that presumably mediates disease-specific risk. Our results can be translated into the development of the SNCA intron 1 CGI region as an attractive therapeutics target for gene therapy in patients who suffer from synucleinopathies due to SNCA dysregulation.

Neuroinflammation is one of the main mechanisms leading to neuronal death and dysfunction in neurodegenerative diseases. The role of microglia as primary mediators of inflammation is unclear in Leigh syndrome (LS) patients. This study aims to elucidate the role of microglia in LS progression by a detailed multipronged analysis of LS neuropathology, mouse and human induced pluripotent stem cells models of Leigh syndrome. We described brain pathology in three cases of Leigh syndrome and performed immunohistochemical staining of autopsy brain of LS patients. We used mouse model of LS (Ndufs4-/-) to study the effect of microglial partial ablation using pharmacologic approach. Genetically modified human induced pluripotent stem cell (iPS) derived neurons and brain organoid with Ndufs4 mutation were used to investigate the neuroinflammation in LS. We reported a novel observation of marked increased in Iba1+ cells with features of activated microglia, in various parts of brain in postmortem neuropathological examinations of three Leigh syndrome patients. Using an Ndufs4-/- mouse model for Leigh syndrome, we showed that partial ablation of microglia by Pexidartinib initiated at the symptom onset improved neurological function and significantly extended lifespan. Ndufs4 mutant LS brain organoid had elevated NLRP3 and IL6 pro-inflammatory pathways. Ndufs4-mutant LS iPSC neurons were more susceptible to glutamate excitotoxicity, which was further potentiated by IL-6. Our findings of LS human brain pathology, Ndufs4-deficient mouse and iPSC models of LS suggest a critical role of activated microglia in the progression of LS encephalopathy. This study suggests a potential clinical application of microglial ablation and immunosuppression during the active phase of Leigh syndrome.

MicroRNAs (miRNAs) that regulate mRNA stability have been linked to amyloid production, tau phosphorylation, and inflammation in Alzheimer's disease (AD). However, whether cerebral miRNA networks are dysregulated during the earliest stages of AD remains underexplored. We performed miRNA expression analysis using frontal cortex tissue harvested from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), amnestic mild cognitive impairment (aMCI, a putative prodromal AD stage), or mild AD. Analysis revealed that the miRNA clusters miR-212/132 and miR-23a/23b were down-regulated in the frontal cortex of aMCI subjects. Both miR-212/132 and miR23a/b are predicted to destabilize the message for sirtuin 1 (sirt1); hence, down-regulation of either miR-212/132 or miR-23a/b in frontal cortex should promote sirt1 mRNA expression in this region. qPCR studies revealed that frontal cortex levels of sirt1 were increased in aMCI. Given the ability of frontal cortex to respond to the onset of dementia by neuronal reorganization, these data suggest that miRNA-mediated up-regulation of the sirt1 pathway represents a compensatory response to the onset of the disease. By contrast, qPCR analysis of inferior temporal cortex, an area affected early in the progression of AD, showed no changes in miR-212/132, miR-23a/b, or sirt1 transcripts in the same aMCI subjects. In vitro mechanistic studies showed that coordinated down-regulation of miR-212 and miR-23a increased sirt1 protein expression and provided neuroprotection from β-amyloid toxicity in human neuronal cells. Taken together, these data suggest a novel miRNA-mediated neuroprotective pathway activated during the progression of AD that may be amenable to therapeutic manipulation.

Object-location memory (OLM) is known to decline with normal aging, a process accelerated in pathological conditions like mild cognitive impairment (MCI). In order to maintain cognitive health and to delay the transition from healthy to pathological conditions, novel strategies are being explored. Tentative evidence suggests that combining cognitive training and anodal transcranial direct current stimulation (atDCS), both reported to induce small and often inconsistent behavioral improvements, could generate larger or more consistent improvements or both, compared to each intervention alone. Here, we explored the combined efficacy of these techniques on OLM. In a subject-blind sham-controlled cross-over design 32 healthy older adults underwent a 3-day visuospatial training paired with either anodal (20 min) or sham (30 s) atDCS (1 mA, temporoparietal). Subjects were asked to learn the correct object-location pairings on a street map, shown over five learning blocks on each training day. Acquisition performance was assessed by accuracy on a given learning block in terms of percentage of correct responses. Training success (performance on last training day) and delayed memory after 1-month were analyzed by mixed model analysis and were controlled for gender, age, education, sequence of stimulation and baseline performance. Exploratory analysis of atDCS effects on within-session (online) and between-session (offline) memory performance were conducted. Moreover, transfer effects on similar trained (visuospatial) and less similar (visuo-constructive, verbal) untrained memory tasks were explored, both immediately after training, and on follow-up. We found that atDCS paired with OLM-training did not enhance success in training or performance in 1-month delayed memory or transfer tasks. In sum, this study did not support the notion that the combined atDCS-training approach improves immediate or delayed OLM in older adults. However, specifics of the experimental design, and a non-optimal timing of atDCS between sessions might have masked beneficial effects and should be more systematically addressed in future studies.

[11C]Verubulin (a.k.a.[11C]MCP-6827), [11C]HD-800 and [11C]colchicine have been developed for imaging microtubules (MTs) with positron emission tomography (PET). The objective of this work was to conduct an in vivo comparison of [11C]verubulin for MT imaging in mouse and rat brain, as well as an in vitro study with this radiotracer in rodent and human Alzheimer's Disease tissue. Our preliminary PET imaging studies of [11C]verubulin in rodents revealed contradictory results between mouse and rat brain uptake under pretreatment conditions. In vitro autoradiography with [11C]verubulin showed an unexpected higher uptake in AD patient tissue compared with healthy controls. We also conducted the first comparative in vivo PET imaging study with [11C]verubulin, [11C]HD-800 and [11C]colchicine in a non-human primate. [11C]Verubulin and [11C]HD-800 require pharmacokinetic modeling and quantification studies to understand the role of how these radiotracers bind to MTs before translation to human use.

Alzheimer's dementia commonly impacts the health of older adults and lacks any preventative therapy. While Alzheimer's dementia risk has a substantial genetic component, the specific molecular mechanisms and neuropathologies triggered by most of the known genetic variants are unclear. Resultantly, they have shown limited influence on drug development portfolios to date. To facilitate our understanding of the consequences of Alzheimer's dementia susceptibility variants, we examined their relationship to a wide range of clinical, molecular and neuropathological features. Because the effect size of individual variants is typically small, we utilized a polygenic (overall) risk approach to identify the global impact of Alzheimer's dementia susceptibility variants. Under this approach, each individual has a polygenic risk score (PRS) that we related to clinical, molecular and neuropathological phenotypes. Applying this approach to 1,272 individuals who came to autopsy from one of two longitudinal aging cohorts, we observed that an individual's PRS was associated with cognitive decline and brain pathologies including beta-amyloid, tau-tangles, hippocampal sclerosis, and TDP-43, MIR132, four proteins including VGF, IGFBP5, and STX1A, and many chromosomal regions decorated with acetylation on histone H3 lysine 9 (H3K9Ac). While excluding the APOE/TOMM40 region (containing the single largest genetic risk factor for late-onset Alzheimer's dementia) in the calculation of the PRS resulted in a slightly weaker association with the molecular signatures, results remained significant. These PRS-associated brain pathologies and molecular signatures appear to mediate genetic risk, as they attenuated the association of the PRS with cognitive decline. Notably, the PRS induced changes in H3K9Ac throughout the genome, implicating it in large-scale chromatin changes. Thus, the PRS for Alzheimer's dementia (AD-PRS) showed effects on diverse clinical, molecular, and pathological systems, ranging from the epigenome to specific proteins. These convergent targets of a large number of genetic risk factors for Alzheimer's dementia will help define the experimental systems and models needed to test therapeutic targets, which are expected to be broadly effective in the aging population that carries diverse genetic risks for Alzheimer's dementia.

Digital pathology is increasingly prominent in neurodegenerative disease research, but variability in immunohistochemical staining intensity between staining batches prevents large-scale comparative studies. Here we provide a statistically rigorous method to account for staining batch effects in a large sample of brain tissue with frontotemporal lobar degeneration with tau inclusions (FTLD-Tau, N = 39) or TDP-43 inclusions (FTLD-TDP, N = 53). We analyzed the relationship between duplicate measurements of digital pathology, i.e., percent area occupied by pathology (%AO) for grey matter (GM) and white matter (WM), from two distinct staining batches. We found a significant difference in duplicate measurements from distinct staining batches in FTLD-Tau (mean difference: GM = 1.13 ± 0.44, WM = 1.28 ± 0.56; p < 0.001) and FTLD-TDP (GM = 0.95 ± 0.66, WM = 0.90 ± 0.77; p < 0.001), and these measurements were linearly related (R-squared [Rsq]: FTLD-Tau GM = 0.92, WM = 0.92; FTLD-TDP GM = 0.75, WM = 0.78; p < 0.001 all). We therefore used linear regression to transform %AO from distinct staining batches into equivalent values. Using a train-test set design, we examined transformation prerequisites (i.e., Rsq) from linear-modeling in training sets, and we applied equivalence factors (i.e., beta, intercept) to independent testing sets to determine transformation outcomes (i.e., intraclass correlation coefficient [ICC]). First, random iterations (×100) of linear regression showed that smaller training sets (N = 12-24), feasible for prospective use, have acceptable transformation prerequisites (mean Rsq: FTLD-Tau ≥0.9; FTLD-TDP ≥0.7). When cross-validated on independent complementary testing sets, in FTLD-Tau, N = 12 training sets resulted in 100% of GM and WM transformations with optimal transformation outcomes (ICC ≥ 0.8), while in FTLD-TDP N = 24 training sets resulted in optimal ICC in testing sets (GM = 72%, WM = 98%). We therefore propose training sets of N = 12 in FTLD-Tau and N = 24 in FTLD-TDP for prospective transformations. Finally, the transformation enabled us to significantly reduce batch-related difference in duplicate measurements in FTLD-Tau (GM/WM: p < 0.001 both) and FTLD-TDP (GM/WM: p < 0.001 both), and to decrease the necessary sample size estimated in a power analysis in FTLD-Tau (GM:-40%; WM: -34%) and FTLD-TDP (GM: -20%; WM: -30%). Finally, we tested generalizability of our approach using a second, open-source, image analysis platform and found similar results. We concluded that a small sample of tissue stained in duplicate can be used to account for pre-analytical variability such as staining batch effects, thereby improving methods for future studies.

The distinct organization of the brain's vasculature ensures the adequate delivery of oxygen and nutrients during development and adulthood. Acute and chronic pathological changes of the vascular system have been implicated in many neurological disorders including stroke and dementia. Here, we describe a fast, automated method that allows the highly reproducible, quantitative assessment of distinct vascular parameters and their changes based on the open source software Fiji (ImageJ). In particular, we developed a practical guide to reliably measure aspects of growth, repair and maturation of the brain's vasculature during development and neurovascular disease in mice and humans. The script can be used to assess the effects of different external factors including pharmacological treatments or disease states. Moreover, the procedure is expandable to blood vessels of other organs and vascular in vitro models.

Alzheimer's disease (AD) is the most prevalent form of dementia, accounting for 60-70% of all dementias. AD is often under-diagnosed and recognized only at a later, more advanced stage, and this delay in diagnosis has been suggested as a contributing factor in the numerous unsuccessful AD treatment trials. Although there is no known cure for AD, early diagnosis is important for disease management and care. A hallmark of AD is the deposition of amyloid-β (Aβ)-containing senile neuritic plaques and neurofibrillary tangles composed of hyperphosporylated tau in the brain. However, current in vivo methods to quantify Aβ in the brain are invasive, requiring radioactive tracers and positron emission tomography. Toward development of alternative methods to assess AD progression, we focus on the retinal manifestation of AD pathology. The retina is an extension of the central nervous system uniquely accessible to light-based, non-invasive ophthalmic imaging. However, earlier studies in human retina indicate that the literature is divided on the presence of Aβ in the AD retina. To help resolve this disparity, this study assessed retinal tissues from neuropathologically confirmed AD cases to determine the regional distribution of Aβ in retinal wholemounts and to inform on future retinal image studies targeting Aβ. Concurrent post-mortem brain tissues were also collected. Neuropathological cortical assessments including neuritic plaque (NP) scores and cerebral amyloid angiopathy (CAA) were correlated with retinal Aβ using immunohistochemistry, confocal microscopy, and quantitative image analysis. Aβ load was compared between AD and control (non-AD) eyes. Our results indicate that levels of intracellular and extracellular Aβ retinal deposits were significantly higher in AD than controls. Mid-peripheral Aβ levels were greater than central retina in both AD and control eyes. In AD retina, higher intracellular Aβ was associated with lower NP score, while higher extracellular Aβ was associated with higher CAA score. Our data support the feasibility of using the retinal tissue to assess ocular Aβ as a surrogate measure of Aβ in the brain of individuals with AD. Specifically, mid-peripheral retina possesses more Aβ deposition than central retina, and thus may be the optimal location for future in vivo ocular imaging.

Alzheimer's disease (AD) is defined by the presence of intraneuronal neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau aggregates as well as extracellular amyloid-beta plaques. The presence and spread of tau pathology through the brain is classified by Braak stages and thought to correlate with the progression of AD. Several in vitro and in vivo studies have examined the ability of tau pathology to move from one neuron to the next, suggesting a "prion-like" spread of tau aggregates may be an underlying cause of Braak tau staging in AD. Using the HEK293 TauRD-P301S-CFP/YFP expressing biosensor cells as a highly sensitive and specific tool to identify the presence of seed competent aggregated tau in brain lysate-i.e., tau aggregates that are capable of recruiting and misfolding monomeric tau-, we detected substantial tau seeding levels in the entorhinal cortex from human cases with only very rare NFTs, suggesting that soluble tau aggregates can exist prior to the development of overt tau pathology. We next looked at tau seeding levels in human brains of varying Braak stages along six regions of the Braak Tau Pathway. Tau seeding levels were detected not only in the brain regions impacted by pathology, but also in the subsequent non-pathology containing region along the Braak pathway. These data imply that pathogenic tau aggregates precede overt tau pathology in a manner that is consistent with transneuronal spread of tau aggregates. We then detected tau seeding in frontal white matter tracts and the optic nerve, two brain regions comprised of axons that contain little to no neuronal cell bodies, implying that tau aggregates can indeed traverse along axons. Finally, we isolated cytosolic and synaptosome fractions along the Braak Tau Pathway from brains of varying Braak stages. Phosphorylated and seed competent tau was significantly enriched in the synaptic fraction of brain regions that did not have extensive cellular tau pathology, further suggesting that aggregated tau seeds move through the human brain along synaptically connected neurons. Together, these data provide further evidence that the spread of tau aggregates through the human brain along synaptically connected networks results in the pathogenesis of human Alzheimer's disease.