The wake- and sleep-modulating neurons of the lateral hypothalamic area demonstrate a differential pattern of degeneration in Alzheimers disease.

BACKGROUND: Sleep-wake dysfunction is an early and common event in Alzheimer's disease (AD). The lateral hypothalamic area (LHA) regulates the sleep and wake cycle through wake-promoting orexinergic neurons (OrxN) and sleep-promoting melanin-concentrating hormone or MCHergic neurons (MCHN). These neurons share close anatomical proximity with functional reciprocity. This study investigated LHA OrxN and MCHN loss patterns in AD individuals. Understanding the degeneration pattern of these neurons will be instrumental in designing potential therapeutics to slow down the disease progression and remediate the sleep-wake dysfunction in AD. METHODS: Postmortem human brain tissue from donors with AD (across progressive stages) and controls were examined using unbiased stereology. Formalin-fixed, celloidin-embedded hypothalamic sections were stained with Orx-A/MCH, p-tau (CP13), and counterstained with gallocyanin. Orx or MCH-positive neurons with or without CP13 inclusions and gallocyanin-stained neurons were considered for stereology counting. Additionally, we extracted RNA from the LHA using conventional techniques. We used customized Neuropathology and Glia nCounter (Nanostring) panels to study gene expression. Wald statistical test was used to compare the groups, and the genes were considered differentially expressed when the p-value was <.05. RESULTS: We observed a progressive decline in OrxN alongside a relative preservation of MCHN. OrxN decreased by 58% (p=0.03) by Braak stages (BB) 1-2 and further declined to 81% (p=0.03) by BB 5-6. Conversely, MCHN demonstrated a non-statistical significant decline (27%, p=0.1088) by BB 6. We observed a progressive increase in differentially expressed genes (DEGs), starting with glial profile changes in BB2. While OrxN loss was observed, Orx-related genes showed upregulation in BB 3-4 compared to BB 0-1. GO and KEGG terms related to neuroinflammatory pathways were mainly enriched. CONCLUSIONS: To date, OrxN loss in the LHA represents the first neuronal population to die preceding the loss of LC neurons. Conversely, MCHN shows resilience to AD p-tau accumulation across Braak stages. The initial loss of OrxN correlates with specific neuroinflammation, glial profile changes, and an overexpression of HCRT, possibly due to hyperexcitation following compensation mechanisms. Interventions preventing OrxN loss and inhibiting p-tau accumulation in the LHA could prevent neuronal loss in AD and, perhaps, the progression of the disease.

Increased NLRP1 mRNA and Protein Expression Suggests Inflammasome Activation in the Dorsolateral Prefrontal and Medial Orbitofrontal Cortex in Schizophrenia.

Schizophrenia is a complex mental condition, with key symptoms marked for diagnosis including delusions, hallucinations, disorganized thinking, reduced emotional expression, and social dysfunction. In the context of major developmental hypotheses of schizophrenia, notably those concerning maternal immune activation and neuroinflammation, we studied NLRP1 expression and content in the postmortem brain tissue of 10 schizophrenia and 10 control subjects. In the medial orbitofrontal cortex (Brodmann's area 11/12) and dorsolateral prefrontal cortex (area 46) from both hemispheres of six schizophrenia subjects, the NLRP1 mRNA expression was significantly higher than in six control brains (p < 0.05). As the expression difference was highest for the medial orbitofrontal cortex in the right hemisphere, we assessed NLRP1-immunoreactive pyramidal neurons in layers III, V, and VI in the medial orbitofrontal cortex in the right hemisphere of seven schizophrenia and five control brains. Compared to controls, we quantified a significantly higher number of NLRP1-positive pyramidal neurons in the schizophrenia brains (p < 0.01), suggesting NLRP1 inflammasome activation in schizophrenia subjects. Layer III pyramidal neuron dysfunction aligns with working memory deficits, while impairments of pyramidal neurons in layers V and VI likely disrupt predictive processing. We propose NLRP1 inflammasome as a potential biomarker and therapeutic target in schizophrenia.

Subcortical Neuronal Correlates of Sleep in Neurodegenerative Diseases.

Importance: Sleep disturbance is common among patients with neurodegenerative diseases. Examining the subcortical neuronal correlates of sleep disturbances is important to understanding the early-stage sleep neurodegenerative phenomena. Objectives: To examine the correlation between the number of important subcortical wake-promoting neurons and clinical sleep phenotypes in patients with Alzheimer disease (AD) or progressive supranuclear palsy (PSP). Design, Setting, and Participants: This longitudinal cohort study enrolled 33 patients with AD, 20 patients with PSP, and 32 healthy individuals from the Memory and Aging Center of the University of California, San Francisco, between August 22, 2008, and December 31, 2020. Participants received electroencephalographic and polysomnographic sleep assessments. Postmortem neuronal analyses of brainstem hypothalamic wake-promoting neurons were performed and were included in the clinicopathological correlation analysis. No eligible participants were excluded from the study. Exposures: Electroencephalographic and polysomnographic assessment of sleep and postmortem immunohistological stereological analysis of 3 wake-promoting nuclei (noradrenergic locus coeruleus [LC], orexinergic lateral hypothalamic area [LHA], and histaminergic tuberomammillary nucleus [TMN]). Main Outcomes and Measures: Nocturnal sleep variables, including total sleep time, sleep maintenance, rapid eye movement (REM) latency, and time spent in REM sleep and stages 1, 2, and 3 of non-REM (NREM1, NREM2, and NREM3, respectively) sleep, and wake after sleep onset. Neurotransmitter, tau, and total neuronal counts of LC, LHA, and TMN. Results: Among 19 patients included in the clinicopathological correlation analysis, the mean (SD) age at death was 70.53 (7.75) years; 10 patients (52.6%) were female; and all patients were White. After adjusting for primary diagnosis, age, sex, and time between sleep analyses and death, greater numbers of LHA and TMN neurons were correlated with decreased homeostatic sleep drive, as observed by less total sleep time (LHA: r = -0.63; P = .009; TMN: r = -0.62; P = .008), lower sleep maintenance (LHA: r = -0.85; P < .001; TMN: r = -0.78; P < .001), and greater percentage of wake after sleep onset (LHA: r = 0.85; P < .001; TMN: r = 0.78; P < .001). In addition, greater numbers of LHA and TMN neurons were correlated with less NREM2 sleep (LHA: r = -0.76; P < .001; TMN: r = -0.73; P < .001). A greater number of TMN neurons was also correlated with less REM sleep (r = -0.61; P = .01). A greater number of LC neurons was mainly correlated with less total sleep time (r = -0.68; P = .008) and greater REM latency (r = 0.71; P = .006). The AD-predominant group had significantly greater sleep drive, including higher total sleep time (mean [SD], 0.49 [1.18] vs -1.09 [1.37]; P = .03), higher sleep maintenance (mean [SD], 0.18 [1.22] vs -1.53 [1.78]; P = .02), and lower percentage of wake after sleep onset during sleep period time (mean [SD], -0.18 [1.20] vs 1.49 [1.72]; P = .02) than the PSP-predominant group based on unbiased k-means clustering and principal component analyses. Conclusions and Relevance: In this cohort study, subcortical wake-promoting neurons were significantly correlated with sleep phenotypes in patients with AD and PSP, suggesting that the loss of wake-promoting neurons among patients with neurodegenerative conditions may disturb the control of sleep-wake homeostasis. These findings suggest that the subcortical system is a primary mechanism associated with sleep disturbances in the early stages of neurodegenerative diseases.

Emergence of distinct and heterogeneous strains of amyloid beta with advanced Alzheimer's disease pathology in Down syndrome.

Amyloid beta (Aß) is thought to play a critical role in the pathogenesis of Alzheimer's disease (AD). Prion-like Aß polymorphs, or "strains", can have varying pathogenicity and may underlie the phenotypic heterogeneity of the disease. In order to develop effective AD therapies, it is critical to identify the strains of Aß that might arise prior to the onset of clinical symptoms and understand how they may change with progressing disease. Down syndrome (DS), as the most common genetic cause of AD, presents promising opportunities to compare such features between early and advanced AD. In this work, we evaluate the neuropathology and Aß strain profile in the post-mortem brain tissues of 210 DS, AD, and control individuals. We assayed the levels of various Aß and tau species and used conformation-sensitive fluorescent probes to detect differences in Aß strains among individuals and populations. We found that these cohorts have some common but also some distinct strains from one another, with the most heterogeneous populations of Aß emerging in subjects with high levels of AD pathology. The emergence of distinct strains in DS at these later stages of disease suggests that the confluence of aging, pathology, and other DS-linked factors may favor conditions that generate strains that are unique from sporadic AD.

A manual multiplex immunofluorescence method for investigating neurodegenerative diseases.

BACKGROUND: Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ is critical to facilitate discovery and validation of pathogenic molecular pathways. Immunostaining methods enable in situ detection of specific targets. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue adhesion and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Furthermore, stripping techniques require antibody-specific optimization, validation, and quality control steps. NEW METHOD: Aiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives based on extensive testing. RESULTS: A protocol using ß-mercaptoethanol and SDS consistently enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue adhesion and antigen integrity in human postmortem tissue. COMPARISON WITH EXISTING METHODS: Our proposed method is optimal for standard lab settings and shows consistent efficacy despite the intricacies of suboptimal human postmortem tissue and the need to strip markers bound to highly aggregated proteins. Additionally, it incorporates quality control steps to validate antibody stripping. CONCLUSIONS: Multiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters during optimization and validation phases of experiments is prudent.

Gene expression profiling of the dorsolateral and medial orbitofrontal cortex in schizophrenia.

Schizophrenia is a complex polygenic disorder of unknown etiology. Over 3,000 candidate genes associated with schizophrenia have been reported, most of which being mentioned only once. Alterations in cognitive processing - working memory, metacognition and mentalization - represent a core feature of schizophrenia, which indicates the involvement of the prefrontal cortex in the pathophysiology of this disorder. Hence we compared the gene expression in postmortem tissue from the left and right dorsolateral prefrontal cortex (DLPFC, Brodmann's area 46), and the medial part of the orbitofrontal cortex (MOFC, Brodmann's area 11/12), in six patients with schizophrenia and six control brains. Although in the past decade several studies performed transcriptome profiling in schizophrenia, this is the first study to investigate both hemispheres, providing new knowledge about possible brain asymmetry at the level of gene expression and its relation to schizophrenia. We found that in the left hemisphere, twelve genes from the DLPFC and eight genes from the MOFC were differentially expressed in patients with schizophrenia compared to controls. In the right hemisphere there was only one gene differentially expressed in the MOFC. We reproduce the involvement of previously reported genes TARDBP and HNRNPC in the pathogenesis of schizophrenia, and report seven novel genes: SART1, KAT7, C1D, NPM1, EVI2A, XGY2, and TTTY15. As the differentially expressed genes only partially overlap with previous studies that analyzed other brain regions, our findings indicate the importance of considering prefrontal cortical regions, especially those in the left hemisphere, for obtaining disease-relevant insights.

De novo expression of dopamine D2 receptors on microglia after stroke.

Dopamine is the predominant catecholamine in the brain and functions as a neurotransmitter. Dopamine is also a potent immune modulator. In this study, we have characterized the expression of dopamine receptors on murine microglia. We found that cultured primary microglia express dopamine D1, D2, D3, D4, and D5 receptors. We specifically focused on the D2 receptor (D2R), a major target of antipsychotic drugs. Whereas D2Rs were strongly expressed on striatal neurons in vivo, we did not detect any D2R expression on resident microglia in the healthy brains of wild-type mice or transgenic mice expressing the green fluorescent protein (GFP) under the control of the Drd2 promoter. However, cerebral ischemia induced the expression of D2R on Iba1-immunoreactive inflammatory cells in the infarct core and penumbra. Notably, D2R expression was confined to CD45(hi) cells, and GFP BM chimeras revealed that D2R was expressed on activated resident microglia as well as on peripherally derived macrophages in the ischemic brain. Importantly, the D2/3R agonist, pramipexole, enhanced the secretion of nitrite by cultured microglia in response to proinflammatory stimuli. Thus, dopamine may serve as a modulator of microglia function during neuroinflammation.

Cerebrospinal fluid markers in differential diagnosis of Alzheimer's disease and vascular dementia.

Alzheimer's disease (AD) and vascular dementia (VaD) are the two most common causes of dementia in old people. They remain difficult to differentiate in practice because of lack of sensitivity and specificity of current clinical diagnostic criteria. Recent molecular and cellular advancements indicate that the use of cerebrospinal fluid markers may improve early detection and differential diagnosis of AD. Our objective in this study was to determine diagnostic accuracy of three cerebrospinal (CSF) markers: total tau protein (t-tau), tau protein phosphorylated on threonine 181 (p-tau181) and tau protein phosphorylated on serine 199 (p-tau199). Using commercially available ELISA kits concentrations of t-tau, p-tau181 and p-tau199 were analyzed in 12 patients with probable AD, 9 patients with VaD and 12 NC subjects. The median levels of all three markers were significantly higher in AD group versus VaD and NC groups. However, when the sensitivity levels were set to 85% or higher, only t-tau and p-tau199 satisfied consensus recommendations (specificity more than 75%) when differentiating AD from VaD. In conclusion, our preliminary data on a small group of selected subjects suggest that the CSF t-tau and p-tau199 levels are useful markers for differentiating AD from VaD.

Abnormal motoneuron migration, differentiation, and axon outgrowth in spinal muscular atrophy.

The role of heterotopic (migratory) motoneurons (HMN) in the pathogenesis of spinal muscular atrophy (SMA) is still controversial. We examined the occurrence and amount of HMN in spinal cord tissue from eight children with SMA (six with SMA-I and two with SMA-II). All affected subjects were carrying a homozygous deletion of exon 7 in the SMN1 gene. Unlike controls, virtually free from HMN, all SMA subjects showed a significant number of HMN at all levels of the spinal cord. Heterotopic neurons were hyperchromatic, located mostly in the ventral white matter and had no axon or dendrites. More than half of the HMN were very undifferentiated, as judged from their lack of immunoreactivity for NeuN and MAP2 proteins. Small numbers of more differentiated heterotopic neurons were also found in the dorsal and lateral white matter region. As confirmed by ultrastructural analysis, in situ end labeling (ISEL) and CD68 immunoreactivity, HMN in the ventral outflow were found to have no synapses, to activate microglial cells, and to eventually die by necrosis. An unbiased quantitative analysis showed a significant negative correlation between age of SMA subjects (a reflection of the clinical severity) and the number of HMN. Subjects who died at older ages had increased number of GFAP-positive astrocytes. Complementing our previous report on motoneuron apoptosis within the ventral horns in SMA, we now propose that abnormal migration, differentiation, and lack of axonal outgrowth may induce motoneuron apoptosis predominantly during early stages, whereas a slower necrosis-like cell death of displaced motoneurons which "escaped" apoptosis characterizes later stages of SMA.