RNA sequencing of olfactory bulb in Parkinson's disease reveals gene alterations associated with olfactory dysfunction.

The olfactory bulb is involved early in the pathophysiology of Parkinson's disease (PD), which is consistent with the early onset of olfactory dysfunction. Identifying the molecular mechanisms through which PD affects the olfactory bulb could lead to a better understanding of the pathophysiology and etiology of olfactory dysfunction in PD. We specifically aimed to assess gene expression changes, affected pathways and co-expression network by whole transcriptomic profiling of the olfactory bulb in subjects with clinicopathologically defined PD. Bulk RNA sequencing was performed on frozen human olfactory bulbs of 20 PD and 20 controls without dementia or any other neurodegenerative disorder, from the Arizona Study of Aging and Neurodegenerative disorders and the Brain and Body Donation Program. Differential expression analysis (19 PD vs 19 controls) revealed 2164 significantly differentially expressed genes (1090 upregulated and 1074 downregulated) in PD. Pathways enriched in downregulated genes included oxidative phosphorylation, olfactory transduction, metabolic pathways, and neurotransmitters synapses while immune and inflammatory responses as well as cellular death related pathways were enriched within upregulated genes. An overrepresentation of microglial and astrocyte-related genes was observed amongst upregulated genes, and excitatory neuron-related genes were overrepresented amongst downregulated genes. Co-expression network analysis revealed significant modules highly correlated with PD and olfactory dysfunction that were found to be involved in the MAPK signaling pathway, cytokine-cytokine receptor interaction, cholinergic synapse, and metabolic pathways. LAIR1 (leukocyte associated immunoglobulin like receptor 1) and PPARA (peroxisome proliferator activated receptor alpha) were identified as hub genes with a high discriminative power between PD and controls reinforcing an important role of neuroinflammation in the olfactory bulb of PD subjects. Olfactory identification test score positively correlated with expression of genes coding for G-coupled protein, glutamatergic, GABAergic, and cholinergic receptor proteins and negatively correlated with genes for proteins expressed in glial olfactory ensheathing cells. In conclusion, this study reveals gene alterations associated with neuroinflammation, neurotransmitter dysfunction, and disruptions of factors involved in the initiation of olfactory transduction signaling that may be involved in PD-related olfactory dysfunction.

Measuring Up: A Comparison of TapeStation 4200 and Bioanalyzer 2100 as Measurement Tools for RNA Quality in Postmortem Human Brain Samples.

The determination of RNA integrity is a critical quality assessment tool for gene expression studies where the experiment's success is highly dependent on the sample quality. Since its introduction in 1999, the gold standard in the scientific community has been the Agilent 2100 Bioanalyzer's RNA integrity number (RIN), which uses a 1-10 value system, from 1 being the most degraded, to 10 being the most intact. In 2015, Agilent launched 4200 TapeStation's RIN equivalent, and reported a strong correlation of r2 of 0.936 and a median error < ±0.4 RIN units. To evaluate this claim, we compared the Agilent 4200 TapeStation's RIN equivalent (RINe) and DV200 to the Agilent 2100 Bioanalyzer's RIN for 183 parallel RNA samples. In our study, using RNA from a total of 183 human postmortem brain samples, we found that the RIN and RINe values only weakly correlate, with an r2 of 0.393 and an average difference of 3.2 RIN units. DV200 also only weakly correlated with RIN (r2 of 0.182) and RINe (r2 of 0.347). Finally, when applying a cut-off value of 6.5 for both metrics, we found that 95.6% of samples passed with RIN, while only 23.5% passed with RINe. Our results suggest that even though RIN (Bioanalyzer) and RINe (TapeStation) use the same 1-10 value system, they should not be used interchangeably, and cut-off values should be calculated independently.

Measuring up: A Comparison of Tapestation 4200 and Bioanalyzer 2100 as Measurement Tools for RNA Quality in Postmortem Human Brain Samples.

Determining RNA integrity is a critical quality assessment tool for gene expression studies where the experiment's success is highly dependent on sample quality. Since its introduction in 1999, the gold standard in the scientific community has been the Agilent 2100 Bioanalyzer's RNA Integrity Number (RIN) which uses a 1-10 value system with 1 being the most degraded to 10 being the most intact. In 2015, Agilent launched the 4200 Tapestation's RIN equivalent and reported a strong correlation of r 2 of 0.936 and median error < ± 0.4 RIN units. To evaluate this claim, we compared the Agilent 4200 Tapestation's RIN equivalent (RINe) and DV200 to the Agilent 2100 Bioanalyzer's RIN for 183 parallel RNA samples. In our study, using RNA from a total of 183 human postmortem brain samples, we found that the RIN and RINe values only weakly correlate with an r 2 of 0.393 and an average difference of 3.2 RIN units. DV200 also only weakly correlated with RIN (r 2 of 0.182) and RINe (r 2 of 0.347). Finally, when applying a cut-off value of 6.5 for both metrics, we found that 95.6% of samples passed with RIN, while only 23.5% passed with RINe. Our results suggest that even though RIN (Bioanalyzer) and RINe (Tapestation) use the same 1-10 value system, they should not be used interchangeably, and cut-off values should be calculated independently.

Cerebral white matter rarefaction has both neurodegenerative and vascular causes and may primarily be a distal axonopathy.

Cerebral white matter rarefaction (CWMR) was considered by Binswanger and Alzheimer to be due to cerebral arteriolosclerosis. Renewed attention came with CT and MR brain imaging, and neuropathological studies finding a high rate of CWMR in Alzheimer disease (AD). The relative contributions of cerebrovascular disease and AD to CWMR are still uncertain. In 1181 autopsies by the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), large-format brain sections were used to grade CWMR and determine its vascular and neurodegenerative correlates. Almost all neurodegenerative diseases had more severe CWMR than the normal control group. Multivariable logistic regression models indicated that Braak neurofibrillary stage was the strongest predictor of CWMR, with additional independently significant predictors including age, cortical and diencephalic lacunar and microinfarcts, body mass index, and female sex. It appears that while AD and cerebrovascular pathology may be additive in causing CWMR, both may be solely capable of this. The typical periventricular pattern suggests that CWMR is primarily a distal axonopathy caused by dysfunction of the cell bodies of long-association corticocortical projection neurons. A consequence of these findings is that CWMR should not be viewed simply as "small vessel disease" or as a pathognomonic indicator of vascular cognitive impairment or vascular dementia.

Hemispheric Asymmetry and Atypical Lobar Progression of Alzheimer-Type Tauopathy.

The spread of neurofibrillary tau pathology in Alzheimer disease (AD) mostly follows a stereotypical pattern of topographical progression but atypical patterns associated with interhemispheric asymmetry have been described. Because histopathological studies that used bilateral sampling are limited, this study aimed to assess interhemispheric tau pathology differences and the presence of topographically atypical cortical spreading patterns. Immunohistochemical staining for detection of tau pathology was performed in 23 regions of interest in 57 autopsy cases comparing bilateral cortical regions and hemispheres. Frequent mild (82% of cases) and occasional moderate (32%) interhemispheric density discrepancies were observed, whereas marked discrepancies were uncommon (7%) and restricted to occipital regions. Left and right hemispheric tau pathology dominance was observed with similar frequencies, except in Braak Stage VI that favored a left dominance. Interhemispheric Braak stage differences were observed in 16% of cases and were more frequent in advanced (IV-VI) versus early (I-III) stages. One atypical lobar topographical pattern in which occipital tau pathology density exceeded frontal lobe scores was identified in 4 cases favoring a left dominant asymmetry. We speculate that asymmetry and atypical topographical progression patterns may be associated with atypical AD clinical presentations and progression characteristics, which should be tested by comprehensive clinicopathological correlations.

SARS-CoV-2 Brain Regional Detection, Histopathology, Gene Expression, and Immunomodulatory Changes in Decedents with COVID-19.

Brains of 42 COVID-19 decedents and 107 non-COVID-19 controls were studied. RT-PCR screening of 16 regions from 20 COVID-19 autopsies found SARS-CoV-2 E gene viral sequences in 7 regions (2.5% of 320 samples), concentrated in 4/20 subjects (20%). Additional screening of olfactory bulb (OB), amygdala (AMY) and entorhinal area for E, N1, N2, RNA-dependent RNA polymerase, and S gene sequences detected one or more of these in OB in 8/21 subjects (38%). It is uncertain whether these RNA sequences represent viable virus. Significant histopathology was limited to 2/42 cases (4.8%), one with a large acute cerebral infarct and one with hemorrhagic encephalitis. Case-control RNAseq in OB and AMY found more than 5000 and 700 differentially expressed genes, respectively, unrelated to RT-PCR results; these involved immune response, neuronal constituents, and olfactory/taste receptor genes. Olfactory marker protein-1 reduction indicated COVID-19-related loss of OB olfactory mucosa afferents. Iba-1-immunoreactive microglia had reduced area fractions in cerebellar cortex and AMY, and cytokine arrays showed generalized downregulation in AMY and upregulation in blood serum in COVID-19 cases. Although OB is a major brain portal for SARS-CoV-2, COVID-19 brain changes are more likely due to blood-borne immune mediators and trans-synaptic gene expression changes arising from OB deafferentation.

Acute Brain Ischemia, Infarction and Hemorrhage in Subjects Dying with or Without Autopsy-Proven Acute Pneumonia.

Stroke is one of the most serious complications of Covid-19 disease but it is still unclear whether stroke is more common with Covid-19 pneumonia as compared to non-Covid-19 pneumonia. We investigated the concurrence rate of autopsy-confirmed acute brain ischemia, acute brain infarction and acute brain hemorrhage with autopsy-proven acute non-Covid pneumonia in consecutive autopsies in the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), a longitudinal clinicopathological study of normal aging and neurodegenerative diseases. Of 691 subjects with a mean age of 83.4 years, acute pneumonia was histopathologically diagnosed in 343 (49.6%); the concurrence rates for histopathologically-confirmed acute ischemia, acute infarction or subacute infarction was 14% and did not differ between pneumonia and non-pneumonia groups while the rates of acute brain hemorrhage were 1.4% and 2.0% of those with or without acute pneumonia, respectively. In comparison, in reviews of Covid-19 publications, reported clinically-determined rates of acute brain infarction range from 0.5% to 20% while rates of acute brain hemorrhage range from 0.13% to 2%. In reviews of Covid-19 autopsy studies, concurrence rates for both acute brain infarction and acute brain hemorrhage average about 10%. Covid-19 pneumonia and non-Covid-19 pneumonia may have similar risks tor concurrent acute brain infarction and acute brain hemorrhage when pneumonia is severe enough to cause death. Additionally, acute brain ischemia, infarction or hemorrhage may not be more common in subjects dying of acute pneumonia than in subjects dying without acute pneumonia.

Olfactory Bulb and Amygdala Gene Expression Changes in Subjects Dying with COVID-19.

In this study we conducted RNA sequencing on two brain regions (olfactory bulb and amygdala) from subjects who died from COVID-19 or who died of other causes. We found several-fold more transcriptional changes in the olfactory bulb than in the amygdala, consistent with our own work and that of others indicating that the olfactory bulb may be the initial and most common brain region infected. To some extent our results converge with pseudotime analysis towards common processes shared between the brain regions, possibly induced by the systemic immune reaction following SARS-CoV-2 infection. Changes in amygdala emphasized upregulation of interferon-related neuroinflammation genes, as well as downregulation of synaptic and other neuronal genes, and may represent the substrate of reported acute and subacute COVID-19 neurological effects. Additionally, and only in olfactory bulb, we observed an increase in angiogenesis and platelet activation genes, possibly associated with microvascular damages induced by neuroinflammation. Through coexpression analysis we identified two key genes (CAMK2B for the synaptic neuronal network and COL1A2 for the angiogenesis/platelet network) that might be interesting potential targets to reverse the effects induced by SARS-CoV-2 infection. Finally, in olfactory bulb we detected an upregulation of olfactory and taste genes, possibly as a compensatory response to functional deafferentation caused by viral entry into primary olfactory sensory neurons. In conclusion, we were able to identify transcriptional profiles and key genes involved in neuroinflammation, neuronal reaction and olfaction induced by direct CNS infection and/or the systemic immune response to SARS-CoV-2 infection.

Vagus Nerve and Stomach Synucleinopathy in Parkinson's Disease, Incidental Lewy Body Disease, and Normal Elderly Subjects: Evidence Against the "Body-First" Hypothesis.

BACKGROUND: Braak and others have proposed that Lewy-type α-synucleinopathy in Parkinson's disease (PD) may arise from an exogenous pathogen that passes across the gastric mucosa and then is retrogradely transported up the vagus nerve to the medulla. OBJECTIVE: We tested this hypothesis by immunohistochemically staining, with a method specific for p-serine 129 α-synuclein (pSyn), stomach and vagus nerve tissue from an autopsy series of 111 normal elderly subjects, 33 with incidental Lewy body disease (ILBD) and 53 with PD. METHODS: Vagus nerve samples were taken adjacent to the carotid artery in the neck. Stomach samples were taken from the gastric body, midway along the greater curvature. Formalin-fixed paraffin-embedded sections were immunohistochemically stained for pSyn, shown to be highly specific and sensitive for α-synuclein pathology. RESULTS: Median disease duration for the PD group was 13 years. In the vagus nerve none of the 111 normal subjects had pSyn in the vagus, while 12/26 ILBD (46%) and 32/36 PD (89%) subjects were pSyn-positive. In the stomach none of the 102 normal subjects had pSyn while 5/30 (17%) ILBD and 42/52 (81%) of PD subjects were pSyn-positive. CONCLUSION: As there was no pSyn in the vagus nerve or stomach of subjects without brain pSyn, these results support initiation of pSyn in the brain. The presence of pSyn in the vagus nerve and stomach of a subset of ILBD cases indicates that synucleinopathy within the peripheral nervous system may occur, within a subset of individuals, at preclinical stages of Lewy body disease.

Increased Risk of Autopsy-Proven Pneumonia with Sex, Season and Neurodegenerative Disease.

There has been a markedly renewed interest in factors associated with pneumonia, a leading cause of death worldwide, due to its frequent concurrence with pandemics of influenza and Covid-19 disease. Reported predisposing factors to both bacterial pneumonia and pandemic viral lower respiratory infections are wintertime occurrence, older age, obesity, pre-existing cardiopulmonary conditions and diabetes. Also implicated are age-related neurodegenerative diseases that cause parkinsonism and dementia. We investigated the prevalence of autopsy-proven pneumonia in the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), a longitudinal clinicopathological study, between the years 2006 and 2019 and before the beginning of the Covid-19 pandemic. Of 691 subjects dying at advanced ages (mean 83.4), pneumonia was diagnosed postmortem in 343 (49.6%). There were 185 subjects without dementia or parkinsonism while clinicopathological diagnoses for the other subjects included 319 with Alzheimer's disease dementia, 127 with idiopathic Parkinson's disease, 72 with dementia with Lewy bodies, 49 with progressive supranuclear palsy and 78 with vascular dementia. Subjects with one or more of these neurodegenerative diseases all had higher pneumonia rates, ranging between 50 and 61%, as compared to those without dementia or parkinsonism (40%). In multivariable logistic regression models, male sex and a non-summer death both had independent contributions (ORs of 1.67 and 1.53) towards the presence of pneumonia at autopsy while the absence of parkinsonism or dementia was a significant negative predictor of pneumonia (OR 0.54). Male sex, dementia and parkinsonism may also be risk factors for Covid-19 pneumonia. The apolipoprotein E4 allele, as well as obesity, chronic obstructive pulmonary disease, diabetes, hypertension, congestive heart failure, cardiomegaly and cigarette smoking history, were not significantly associated with pneumonia, in contradistinction to what has been reported for Covid-19 disease.

Mapping of SARS-CoV-2 Brain Invasion and Histopathology in COVID-19 Disease.

The coronavirus SARS-CoV-2 (SCV2) causes acute respiratory distress, termed COVID-19 disease, with substantial morbidity and mortality. As SCV2 is related to previously-studied coronaviruses that have been shown to have the capability for brain invasion, it seems likely that SCV2 may be able to do so as well. To date, although there have been many clinical and autopsy-based reports that describe a broad range of SCV2-associated neurological conditions, it is unclear what fraction of these have been due to direct CNS invasion versus indirect effects caused by systemic reactions to critical illness. Still critically lacking is a comprehensive tissue-based survey of the CNS presence and specific neuropathology of SCV2 in humans. We conducted an extensive neuroanatomical survey of RT-PCR-detected SCV2 in 16 brain regions from 20 subjects who died of COVID-19 disease. Targeted areas were those with cranial nerve nuclei, including the olfactory bulb, medullary dorsal motor nucleus of the vagus nerve and the pontine trigeminal nerve nuclei, as well as areas possibly exposed to hematogenous entry, including the choroid plexus, leptomeninges, median eminence of the hypothalamus and area postrema of the medulla. Subjects ranged in age from 38 to 97 (mean 77) with 9 females and 11 males. Most subjects had typical age-related neuropathological findings. Two subjects had severe neuropathology, one with a large acute cerebral infarction and one with hemorrhagic encephalitis, that was unequivocally related to their COVID-19 disease while most of the 18 other subjects had non-specific histopathology including focal ß-amyloid precursor protein white matter immunoreactivity and sparse perivascular mononuclear cell cuffing. Four subjects (20%) had SCV2 RNA in one or more brain regions including the olfactory bulb, amygdala, entorhinal area, temporal and frontal neocortex, dorsal medulla and leptomeninges. The subject with encephalitis was SCV2-positive in a histopathologically-affected area, the entorhinal cortex, while the subject with the large acute cerebral infarct was SCV2-negative in all brain regions. Like other human coronaviruses, SCV2 can inflict acute neuropathology in susceptible patients. Much remains to be understood, including what viral and host factors influence SCV2 brain invasion and whether it is cleared from the brain subsequent to the acute illness.

Human Autopsy-Derived Scalp Fibroblast Biobanking for Age-Related Neurodegenerative Disease Research.

The Arizona Study of Aging and Neurodegenerative Disorders/Brain and Body Donation Program at Banner Sun Health Research Institute (BSHRI) is a longitudinal clinicopathological study with a current enrollment of more than 900 living subjects for aging and neurodegenerative disease research. Annual clinical assessments are done by cognitive and movement neurologists and neuropsychologists. Brain and body tissues are collected at a median postmortem interval of 3.0 h for neuropathological diagnosis and banking. Since 2018, the program has undertaken banking of scalp fibroblasts derived from neuropathologically characterized donors with Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. Here, we describe the procedure development and cell characteristics from 14 male and 15 female donors (mean ± SD of age: 83.6 ± 12.2). Fibroblasts from explant cultures were banked at passage 3. The results of mRNA analysis showed positive expression of fibroblast activation protein, vimentin, fibronectin, and THY1 cell surface antigen. We also demonstrated that the banked fibroblasts from a postmortem elderly donor were successfully reprogramed to human-induced pluripotent stem cells (hiPSCs). Taken together, we have demonstrated the successful establishment of a human autopsy-derived fibroblast banking program. The cryogenically preserved cells are available for request at the program website of the BSHRI.

Severe hyposmia distinguishes neuropathologically confirmed dementia with Lewy bodies from Alzheimer's disease dementia.

Many subjects with neuropathologically-confirmed dementia with Lewy bodies (DLB) are never diagnosed during life, instead being categorized as Alzheimer's disease dementia (ADD) or unspecified dementia. Unrecognized DLB therefore is a critical impediment to clinical studies and treatment trials of both ADD and DLB. There are studies that suggest that olfactory function tests may be able to distinguish DLB from ADD, but few of these had neuropathological confirmation of diagnosis. We compared University of Pennsylvania Smell Identification Test (UPSIT) results in 257 subjects that went on to autopsy and neuropathological examination. Consensus clinicopathological diagnostic criteria were used to define ADD and DLB, as well as Parkinson's disease with dementia (PDD), with (PDD+AD) or without (PDD-AD) concurrent AD; a group with ADD and Lewy body disease (LBD) not meeting criteria for DLB (ADLB) and a clinically normal control group were also included. The subjects with DLB, PDD+AD and PDD-AD all had lower (one-way ANOVA p < 0.0001, pairwise Bonferroni p < 0.05) first and mean UPSIT scores than the ADD, ADLB or control groups. For DLB subjects with first and mean UPSIT scores less than 20 and 17, respectively, Firth logistic regression analysis, adjusted for age, gender and mean MMSE score, conferred statistically significant odds ratios of 17.5 and 18.0 for the diagnosis, vs ADD. For other group comparisons (PDD+AD and PDD-AD vs ADD) and UPSIT cutoffs of 17, the same analyses resulted in odds ratios ranging from 16.3 to 31.6 (p < 0.0001). To our knowledge, this is the largest study to date comparing olfactory function in subjects with neuropathologically-confirmed LBD and ADD. Olfactory function testing may be a convenient and inexpensive strategy for enriching dementia studies or clinical trials with DLB subjects, or conversely, reducing the inclusion of DLB subjects in ADD studies or trials.

Faster cognitive decline in dementia due to Alzheimer disease with clinically undiagnosed Lewy body disease.

BACKGROUND: Neuropathology has demonstrated a high rate of comorbid pathology in dementia due to Alzheimer's disease (ADD). The most common major comorbidity is Lewy body disease (LBD), either as dementia with Lewy bodies (AD-DLB) or Alzheimer's disease with Lewy bodies (AD-LB), the latter representing subjects with ADD and LBD not meeting neuropathological distribution and density thresholds for DLB. Although it has been established that ADD subjects with undifferentiated LBD have a more rapid cognitive decline than those with ADD alone, it is still unknown whether AD-LB subjects, who represent the majority of LBD and approximately one-third of all those with ADD, have a different clinical course. METHODS: Subjects with dementia included those with "pure" ADD (n = 137), AD-DLB (n = 64) and AD-LB (n = 114), all with two or more complete Mini Mental State Examinations (MMSE) and a full neuropathological examination. RESULTS: Linear mixed models assessing MMSE change showed that the AD-LB group had significantly greater decline compared to the ADD group (ß = -0.69, 95% CI: -1.05, -0.33, p<0.001) while the AD-DLB group did not (ß = -0.30, 95% CI: -0.73, 0.14, p = 0.18). Of those with AD-DLB and AD-LB, only 66% and 2.1%, respectively, had been diagnosed with LBD at any point during their clinical course. Compared with clinically-diagnosed AD-DLB subjects, those that were clinically undetected had significantly lower prevalences of parkinsonism (p = 0.046), visual hallucinations (p = 0.0008) and dream enactment behavior (0.013). CONCLUSIONS: The probable cause of LBD clinical detection failure is the lack of a sufficient set of characteristic core clinical features. Core DLB clinical features were not more common in AD-LB as compared to ADD. Clinical identification of ADD with LBD would allow stratified analyses of ADD clinical trials, potentially improving the probability of trial success.

Impaired hepatic amyloid-beta degradation in Alzheimer's disease.

Extensive research strongly suggests that amyloid beta (Aß) aggregates in the brain have a central role in Alzheimer's disease (AD) pathogenesis. Pathological Aß deposition is likely due to an altered balance between overproduction and elimination. Rodent studies have suggested that the liver has a major role in Aß degradation. It is possible alterations of liver function could affect brain Aß levels through changes in blood Aß concentration. In this study, we hypothesized hepatic Aß degradation to be impaired in AD subjects. To test our hypothesis, an Aß degradation assay was developed using synthetic fluorescein-labeled Aß40 and Aß42 spiked into human liver homogenates. Aß degradation rates were lower in AD-derived homogenates as compared with those from non-demented (ND) control subjects, even after accounting for such covariates as age, sex, and APOE genotype. The protein expression of potential Aß-degrading enzymes were also examined. Neprilysin levels were not different in AD liver samples, while cathepsin D and insulin-degrading enzyme were significantly altered in AD subjects. The results support the possibility that impaired hepatic Aß degradation could be a factor contributing to increased brain Aß accumulation and AD.

Immunohistochemical Method and Histopathology Judging for the Systemic Synuclein Sampling Study (S4).

Immunohistochemical (IHC) α-synuclein (Asyn) pathology in peripheral biopsies may be a biomarker of Parkinson disease (PD). The multi-center Systemic Synuclein Sampling Study (S4) is evaluating IHC Asyn pathology within skin, colon and submandibular gland biopsies from 60 PD and 20 control subjects. Asyn pathology is being evaluated by a blinded panel of specially trained neuropathologists. Preliminary work assessed 2 candidate immunoperoxidase methods using a set of PD and control autopsy-derived sections from formalin-fixed, paraffin-embedded blocks of the 3 tissues. Both methods had 100% specificity; one, utilizing the 5C12 monoclonal antibody, was more sensitive in skin (67% vs 33%), and was chosen for further use in S4. Four trainee neuropathologists were trained to perform S4 histopathology readings; in subsequent testing, their scoring was compared to that of the trainer neuropathologist on both glass slides and digital images. Specificity and sensitivity were both close to 100% with all readers in all tissue types on both glass slides and digital images except for skin, where sensitivity averaged 75% with digital images and 83.5% with glass slides. Semiquantitative (0-3) density score agreement between trainees and trainer averaged 67% for glass slides and 62% for digital images.