Optogenetics has provided a revolutionary approach to dissecting biological phenomena. However, the generation and use of optically active GPCRs in these contexts is limited and it is unclear how well an opsin-chimera GPCR might mimic endogenous receptor activity. Here we show that a chimeric rhodopsin/β2 adrenergic receptor (opto-β2AR) is similar in dynamics to endogenous β2AR in terms of: cAMP generation, MAP kinase activation and receptor internalization. In addition, we develop and characterize a novel toolset of optically active, functionally selective GPCRs that can bias intracellular signalling cascades towards either G-protein or arrestin-mediated cAMP and MAP kinase pathways. Finally, we show how photoactivation of opto-β2AR in vivo modulates neuronal activity and induces anxiety-like behavioural states in both fiber-tethered and wireless, freely moving animals when expressed in brain regions known to contain β2ARs. These new GPCR approaches enhance the utility of optogenetics and allow for discrete spatiotemporal control of GPCR signalling in vitro and in vivo.

Although several lines of evidence indicate that glutamate is a neurotransmitter in primary afferent terminals, controversies exist on the proportion and types of such terminals that release glutamate. In the present study quantitative analysis of immunogold labelling was used to assess the presence of glutamate-like immunoreactivity in primary afferent terminals in laminae I-V of the rat spinal cord dorsal horn. Anterograde transport of choleragenoid-horseradish peroxidase from a spinal ganglion and tetramethyl benzidine histochemistry were used to identify primary afferent terminals in laminae I and III-V. Presumed C-fibre terminals in lamina II were identified on morphological criteria (dense sinusoid axon terminals). Primary afferent terminals in all dorsal horn laminae displayed significantly higher levels of glutamate-like immunoreactivity than pleomorphic vesicle-containing profiles in laminae III-IV and large neuronal cell bodies in laminae III-V. The density of gold particles over primary afferent terminals also significantly exceeded the average density of gold particles over laminae II and III-IV. The highest densities of gold particles were present over dense sinusoid axon terminals in lamina II. These findings suggest that glutamate, alone or in combination with other neuroactive compounds, is involved in the transfer of all sensory modalities from primary afferent fibres to dorsal horn neurons.

Over the course of most common neurodegenerative diseases the amygdala accumulates pathologically misfolded proteins. Misfolding of 1 protein in aged brains often is accompanied by the misfolding of other proteins, suggesting synergistic mechanisms. The multiplicity of pathogenic processes in human amygdalae has potentially important implications for the pathogenesis of Alzheimer disease, Lewy body diseases, chronic traumatic encephalopathy, primary age-related tauopathy, and hippocampal sclerosis, and for the biomarkers used to diagnose those diseases. Converging data indicate that the amygdala may represent a preferential locus for a pivotal transition from a relatively benign clinical condition to a more aggressive disease wherein multiple protein species are misfolded. Thus, understanding of amygdalar pathobiology may yield insights relevant to diagnoses and therapies; it is, however, a complex and imperfectly defined brain region. Here, we review aspects of amygdalar anatomy, connectivity, vasculature, and pathologic involvement in neurodegenerative diseases with supporting data from the University of Kentucky Alzheimer's Disease Center autopsy cohort. Immunohistochemical staining of amygdalae for Aβ, Tau, α-synuclein, and TDP-43 highlight the often-coexisting pathologies. We suggest that the amygdala may represent an "incubator" for misfolded proteins and that it is possible that misfolded amygdalar protein species are yet to be discovered.

Quantitative neuropathologic methods provide information that is important for both research and clinical applications. The technological advancement of digital pathology and image analysis offers new solutions to enable valid quantification of pathological severity that is reproducible between raters regardless of experience. Using an Aperio ScanScope XT and its accompanying image analysis software, we designed algorithms for quantitation of amyloid and tau pathologies on 65 β-amyloid (6F/3D antibody) and 48 phospho-tau (PHF-1)-immunostained sections of human temporal neocortex. Quantitative digital pathologic data were compared with manual pathology counts. There were excellent correlations between manually counted and digitally analyzed neuropathologic parameters (R2 values 0.56-0.72). Data were highly reproducible among 3 participants with varying degrees of expertise in neuropathology (Intra-class correlation coefficient values >0.910). Digital quantification also provided additional parameters, including average plaque area, which show statistically significant differences when samples are stratified according to APOE allele status (average plaque area 380.9 μm2 in ApoE ε4 carriers vs. 274.4 μm2 for non-carriers, p < 0.001). Thus, digital pathology offers a rigorous and reproducible method for quantifying AD neuropathologic changes and may provide additional insight into morphologic characteristics that were previously more challenging to assess due to technical limitations.

Although several studies have applied single-cell approaches to explore gene expression changes in aged brains, they were limited by the relatively shallow sampling of brain cell populations, and thus may have failed to capture aspects of the molecular signatures and dynamics of rare cell types associated with aging and diseases. Here, we set out to investigate the age-dependent dynamics of transcription and chromatin accessibility across diverse brain cell types. With EasySci, an extensively improved single-cell combinatorial indexing strategy, we profiled ~1.5 million single-cell transcriptomes and ~400,000 single-cell chromatin accessibility profiles across mouse brains spanning different ages, genotypes, and both sexes. With a novel computational framework designed for characterizing cellular subtypes based on the expression of both genes and exons, we identified > 300 cell subtypes and deciphered the underlying molecular programs and spatial locations of rare cell types (e.g., pinealocytes, tanycytes) and subtypes. Leveraging these data, we generate a global readout of age-dependent cell population dynamics with high cellular subtype resolution, providing insights into cell types that expand (e.g., rare astrocytes and vascular leptomeningeal cells in the olfactory bulb, reactive microglia and oligodendrocytes) or are depleted (e.g., neuronal progenitors, neuroblasts, committed oligodendrocyte precursors) as age progresses. Furthermore, we explored cell-type-specific responses to genetic perturbations associated with Alzheimer's disease (AD) and identify rare cell types depleted (e.g., mt-Cytb+, mt-Rnr2+ choroid plexus epithelial cells) or enriched (e.g., Col25a1+, Ndrg1+ interbrain and midbrain neurons) in both AD models. Key findings are consistent between males and females, validated across the transcriptome, chromatin accessibility, and spatial analyses. Finally, we profiled a total of 118,240 single-nuclei transcriptomes from twenty-four human brain samples derived from control and AD patients, revealing highly cell-type-specific and region-specific gene expression changes associated with AD pathogenesis. Critical AD-associated gene signatures were validated in both human and mice. In summary, these data comprise a rich resource for exploring cell-type-specific dynamics and the underlying molecular mechanisms in both normal and pathological mammalian aging.

Hippocampal sclerosis of aging (HS-Aging) is a neurodegenerative disease that mimics Alzheimer disease (AD) clinically and has a prevalence rivaling AD in advanced age. Whereas clinical biomarkers are not yet optimized, HS-Aging has distinctive pathological features that distinguish it from other diseases with “hippocampal sclerosis” pathology, such as epilepsy, cerebrovascular perturbations, and frontotemporal lobar degeneration. By definition, HS-Aging brains show neuronal cell loss and gliosis in the hippocampal formation out of proportion to AD-type pathology; it is strongly associated with aberrant TDP-43 pathology and arteriolosclerosis. Here, we describe 2 cases of “segmental” HS-Aging in which “sclerosis” in the hippocampus was evident only in a subset of brain sections by hematoxylin and eosin (H&E) stain. In these cases, TDP-43 pathology was more widespread on immunostained sections than the neuronal cell loss and gliosis seen in H&E stains. The 2 patients were cognitively intact at baseline and were tracked longitudinally over a decade using cognitive studies with at least 1 neuroimaging scan. We discuss the relevant HS-Aging literature, which indicates the need for a clearer consensus-based delineation of “hippocampal sclerosis” and TDP-43 pathologies in aged subjects.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

Introduction: Clinically, detection of disease-causing pathology associated with Alzheimer's disease (AD) and vascular contributions to cognitive impairment and dementia (VCID) is limited to magnetic resonance imaging and positron emission tomography scans, which are expensive and not widely accessible. Here, we assess angiogenic, inflammatory, and AD-related plasma biomarkers to determine their relationships with human post mortem neuropathology.Method: Plasma samples were analyzed using a digital immunoassay and pathological evaluation was performed by University of Kentucky Alzheimer's Disease Research Center neuropathologists. The association of plasma markers with neuropathology was estimated via proportional odds and logistic regressions adjusted for age.Results: Included cases (N = 90) showed increased tau/amyloid beta (Aβ)42 ratio, glial fibrillary acidic protein (GFAP), vascular endothelial growth factor A (VEGF-A), and placental growth factor (PlGF) were positively associated with higher level of AD neuropathological change, while higher Aβ42/Aβ40 ratio was inversely associated. Higher PlGF, VEGF-A, and interleukin 6 were inversely associated with chronic cerebrovascular disease, while Aβ42/Aβ40 ratio was positively associated. Discussion: Our results provide support for the continued study of plasma biomarkers as a clinical screening tool for AD and VCID pathology.