Original Papers

Gilbert Georg Klamminger, Karoline Klein, Laurent Mombaerts, Finn Jelke, Giulia Mirizzi, Rédouane Slimani, Andreas Husch, Michel Mittelbronn, Frank Hertel, Felix Bruno Kleine Borgmann
Free Neuropathol 2: 26 (2021)
Objective and Methods: Timely discrimination between primary CNS lymphoma (PCNSL) and glioblastoma is crucial for diagnostics and therapy, but most importantly also determines the intraoperative surgical course. Advanced radiological methods allow this to a certain extent but ultimately, biopsy is still necessary for final diagnosis. As an upcoming method that enables tissue analysis by tracking changes in the vibrational state of molecules via inelastic scattered photons, we used Raman Spectroscopy (RS) as a label free method to examine specimens of both tumor entities intraoperatively, as well as postoperatively in formalin fixed paraffin embedded (FFPE) samples.

Results: We applied and compared statistical performance of linear and nonlinear machine learning algorithms (Logistic Regression, Random Forest and XGBoost), and found that Random Forest classification distinguished the two tumor entities with a balanced accuracy of 82,4% in intraoperative tissue condition and with 94% using measurements of distinct tumor areas on FFPE tissue. Taking a deeper insight into the spectral properties of the tumor entities, we describe different tumor-specific Raman shifts of interest for classification.

Conclusions: Due to our findings, we propose RS as an additional tool for fast and non-destructive, perioperative tumor tissue discrimination, which may augment treatment options at an early stage. RS may further serve as a useful additional tool for neuropathological diagnostics with little requirements for tissue integrity.
Jonathan D. Cherry, Filisia Agus, Erin Dixon, Bertrand Huber, Victor E. Alvarez, Jesse Mez, Ann C. McKee, Adam Labadorf, Thor D. Stein
Free Neuropathol 2: 21 (2021)
Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative tauopathy found in individuals with a history of repetitive head impacts (RHI). Previous work has demonstrated that neuroinflammation is involved in CTE pathogenesis, however, the specific inflammatory mechanisms are still unclear. Here, using RNA-sequencing and gene set enrichment analysis (GSEA), we investigated the genetic changes found in tissue taken from the region CTE pathology is first found, the cortical sulcus, and compared it to neighboring gryal crest tissue to identify what pathways were directly related to initial hyperphosphorylated tau (p-tau) deposition. 21 cases were chosen for analysis: 6 cases had no exposure to RHI or presence of neurodegenerative disease (Control), 5 cases had exposure to RHI but no presence of neurodegenerative disease (RHI), and 10 cases had exposure to RHI and low stage CTE (CTE). Two sets of genes were identified: genes that changed in both the sulcus and crest and genes that changed specifically in the sulcus relative to the crest. When examining genes that changed in both the sulcus and crest, GSEA demonstrated an increase in immune related processes and a decrease in neuronal processes in RHI and CTE groups. Sulcal specific alterations were observed to be driven by three mechanisms: anatomy, RHI, or p-tau. First, we observed consistent sulcal specific alterations in immune, extracellular matrix, vascular, neuronal, and endocytosis/exocytosis categories across all groups, suggesting the sulcus has a unique molecular signature compared to the neighboring crest independent of pathology. Second, individuals with a history of RHI demonstrated impairment in metabolic and mitochondrial related processes. Finally, in individuals with CTE, we observed impairment of immune and phagocytic related processes. Overall, this work provides the first observation of biological processes specifically altered in the sulcus that could be directly implicated in CTE pathogenesis and provide novel targets for biomarkers and therapies.
Fatma E. El-Khouly, Rianne Haumann, Marjolein Breur, Sophie E.M. Veldhuijzen van Zanten, Gertjan J.L. Kaspers, N. Harry Hendrikse, Esther Hulleman, Dannis G. van Vuurden, Marianna Bugiani
Free Neuropathol 2: 17 (2021)
Aims: Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a median overall survival of eleven months. Lack of chemotherapy efficacy may be related to an intact blood-brain barrier (BBB). In this study we aim to investigate the neurovascular unit (NVU) in DIPG patients.

Methods: DIPG biopsy (n = 4) and autopsy samples (n = 6) and age-matched healthy pons samples (n = 20) were immunohistochemically investigated for plasma protein extravasation, and the expression of tight junction proteins claudin-5 and zonula occludens-1 (ZO-1), basement membrane component laminin, pericyte marker PDGFR-β, and efflux transporters P-gp and BCRP. The mean vascular density and diameter were also assessed.

Results: DIPGs show a heterogeneity in cell morphology and evidence of BBB leakage. Both in tumor biopsy and autopsy samples, expression of claudin-5, ZO-1, laminin, PDGFR-β and P-gp was reduced compared to healthy pontine tissues. In DIPG autopsy samples, vascular density was lower compared to healthy pons. The density of small vessels (<10 µm) was significantly lower (P<0.001), whereas the density of large vessels (≥10 µm) did not differ between groups (P = 0.404). The median vascular diameter was not significantly different: 6.21 µm in DIPG autopsy samples (range 2.25-94.85 µm), and 6.26 µm in controls (range 1.17-264.77 µm).

Conclusion: Our study demonstrates evidence of structural changes in the NVU in DIPG patients, both in biopsy and autopsy samples, as well as a reduced vascular density in end-stage disease. Adding such a biological perspective may help to better direct future treatment choices for DIPG patients.
Stefan Grathwohl, Emmanuel Quansah, Nazia Maroof, Jennifer A. Steiner, Liz Spycher, Fethalla Benmansour, Gonzalo Duran-Pacheco, Juliane Siebourg-Polster, Krisztina Oroszlan-Szovik, Helga Remy, Markus Haenggi, Marc Stawiski, Matthias Selhausen, Pierre Mailver, Andreas Wolfert, Thomas Emrich, Zachary Madaj, Arel Su, Martha L. Escobar Galvis, Christoph Mueller, Annika Herrmann, Patrik Brundin, Markus Britschgi
Free Neuropathol 2: 13 (2021)
Background: In some people with Parkinson’s disease (PD), a-synuclein (αSyn) accumulation may begin in the enteric nervous system (ENS) decades before development of brain pathology and disease diagnosis.

Objective: To determine how different types and severity of intestinal inflammation could trigger αSyn accumulation in the ENS and the subsequent development of αSyn brain pathology.

Methods: We assessed the effects of modulating short- and long-term experimental colitis on αSyn accumulation in the gut of αSyn transgenic and wild type mice by immunostaining and gene expression analysis. To determine the long-term effect on the brain, we induced dextran sulfate sodium (DSS) colitis in young αSyn transgenic mice and aged them under normal conditions up to 9 or 21 months before tissue analyses.

Results: A single strong or sustained mild DSS colitis triggered αSyn accumulation in the submucosal plexus of wild type and αSyn transgenic mice, while short-term mild DSS colitis or inflammation induced by lipopolysaccharide did not have such an effect. Genetic and pharmacological modulation of macrophage-associated pathways modulated the severity of enteric αSyn. Remarkably, experimental colitis at three months of age exacerbated the accumulation of aggregated phospho-Serine 129 αSyn in the midbrain (including the substantia nigra), in 21- but not 9-month-old αSyn transgenic mice. This increase in midbrain αSyn accumulation is accompanied by the loss of tyrosine hydroxylase-immunoreactive nigral neurons.

Conclusions: Our data suggest that specific types and severity of intestinal inflammation, mediated by monocyte/macrophage signaling, could play a critical role in the initiation and progression of PD.
Wouter Peelaerts, Liza Bergkvist, Sonia George, Michaela Johnson, Lindsay Meyerdirk, Emily Schulz, Jennifer A. Steiner, Zachary Madaj, Jiyan Ma, Katelyn Becker, K. Peter R. Nilsson, Jerry R. Colca, Patrik Brundin
Free Neuropathol 1: 33 (2020)
Epidemiological studies suggest a link between type-2 diabetes and Parkinson’s disease (PD) risk. Treatment of type-2 diabetes with insulin sensitizing drugs lowers the risk of PD. We previously showed that the insulin sensitizing drug, MSDC-0160, ameliorates pathogenesis in some animal models of PD. MSDC-0160 reversibly binds the mitochondrial pyruvate carrier (MPC) protein complex, which has an anti-inflammatory effect and restores metabolic deficits. Since PD is characterized by the deposition of α-synuclein (αSyn), we hypothesized that inhibiting the MPC might directly inhibit αSyn aggregation in vivo in mammals. To answer if modulation of MPC can reduce the development of αSyn assemblies, and reduce neurodegeneration, we treated two chronic and progressive mouse models; a viral vector-based αSyn overexpressing model and a pre-formed fibril (PFF) αSyn seeding model with MSDC-0160. These two models present distinct types of αSyn pathology but lack inflammatory or autophagy deficits. Contrary to our hypothesis, we found that a modulation of MPC in these models did not reduce the accumulation of αSyn aggregates or mitigate neurotoxicity. Instead, MSDC-0160 changed the post-translational modification and aggregation features of αSyn. These results are consistent with the lack of a direct effect of MPC modulation on synuclein clearance in these models.
Antigoni Manousopoulou, Ho Ming Yuen, Matt MacGregor Sharp, Satoshi Saito, Roxana Aldea, Norman Mazer, Spiros D Garbis, Roxana O Carare
Free Neuropathol 1: 28 (2020)
Aims: Cerebral amyloid angiopathy (CAA) is the accumulation of amyloid beta (Aβ) in the walls of cerebral arterioles, arteries and capillaries. Changes in the white matter in CAA are observed as hyperintensities and dilated perivascular spaces on MRI suggesting impairment of fluid drainage but the pathophysiology behind these changes is poorly understood. We tested the hypothesis that proteins associated with clearance of Aβ peptides are upregulated in the white matter in cases of CAA.

Methods: In this study, we compare the quantitative proteomic profile of white matter from post-mortem brains of patients with CAA and age-matched controls in order to gain insight into the cellular processes and key molecules involved in the pathophysiology of CAA.

Results: Our proteomic analysis resulted in the profiling of 3,734 proteins (peptide FDR p<0.05). Of these, 189 were differentially expressed in CAA vs. control. Bioinformatics analysis of these proteins showed significant enrichment of proteins related to cell adhesion | cell-matrix interaction, mitochondrial dysfunction and hypoxia. Upregulated proteins in CAA included EMILIN2, COL4A2, TLN1, CLU, HSPG2. Downregulated proteins included DSP, IDE, HBG1.

Conclusions: The present study reports an in-depth quantitative proteomic profiling of white matter from patients with CAA, highlighting extracellular matrix proteins and clusterin as key molecules in the pathophysiology of white matter changes in cases of CAA.
Murad Alturkustani, Qi Zhang, Basma AlYamany, Lee-Cyn Ang
Free Neuropathol 1: 27 (2020)
Two different pathological mechanisms have been suggested to underlie adult-onset leukoencephalopathy with axonal spheroids (ALAS). Pathological studies have suggested that ALAS involves primary axonopathy with secondary demyelination. However, the identification of mutations in Colony Stimulating Factor 1 Receptor (CSF1R), important for microglial survival, has suggested that ALAS is a microgliopathy. This study examines the correlation between microglial changes and axonopathy in ALAS. A total of 6 ALAS cases were studied. White matter lesions were classified into three evolving stages: 1) numerous axonal spheroids among well-myelinated fibers; 2) moderate loss of myelinated fibers with or without axonal spheroids; and 3) a leukodystrophy-like pattern of severe confluent axonal and myelin loss. Axonal spheroids and ramified microglia were semi-quantified and the lesions were assigned a score of 0–3. We found a strong correlation between the preponderance of axonal spheroids and ramified microglial loss. All areas with a predominance of axonal spheroids showed a near-complete absence of ramified microglia, which was also apparent in small cortical and white matter lesions. In contrast, some areas with no ramified microglia showed no axonal pathology. Our findings support the suggestion that ramified microglia loss precedes axonal spheroids formation. This observation will help to better understand the pathogenesis of ALAS and suggests a protective role of microglia.
Elaine Y. Liu, Jenny Russ, Edward B. Lee
Free Neuropathol 1: 23 (2020)
A hexanucleotide G4C2 repeat expansion in C9orf72 is the most common genetic cause of familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). The mutation is associated with a reduction of C9orf72 protein and accumulation of toxic RNA and dipeptide repeat aggregates. The accumulation of toxic RNA has been proposed to sequester RNA binding proteins thereby altering RNA processing, consistent with previous transcriptome studies that have shown that the C9orf72 repeat expansion is linked to abundant splicing alterations and transcriptome changes. Here, we used a subcellular fractionation method and FACS to enrich for neuronal nuclei from C9orf72 repeat expanded post-mortem human ALS/FTD brains, and to remove neuronal nuclei with TDP-43 pathology which are observed in nearly all symptomatic C9orf72 repeat expanded cases. We show that the C9orf72 expansion is associated with relatively mild gene expression changes. Dysregulated genes were enriched for vesicle transport pathways, which is consistent with the known functions of C9orf72 protein. Further analysis suggests that the C9orf72 transcriptome is not driven by toxic RNA but is rather shaped by the depletion of pathologic TDP-43 nuclei and the loss of C9orf72 expression. These findings argue against RNA binding protein sequestration in neurons as a major contributor to C9orf72 mediated toxicity.
Patrick Jarmo Paasila, Danielle S Davies, Greg T Sutherland, Claire Goldsbury
Free Neuropathol 1: 20 (2020)
Alzheimer’s disease (AD) is a late-onset disease that has proved difficult to model. Microglia are implicated in AD, but reports vary on precisely when and how in the sequence of pathological changes they become involved. Here, post-mortem human tissue from two differentially affected regions of the AD brain and from non-demented individuals with a high load of AD-type pathology (high pathology controls) was used to model the disease time course in order to determine how microglial activation relates temporally to the deposition of hallmark amyloid-β (Aβ) and hyperphosphorylated microtubule associated protein tau pathology. Immunofluorescence against the pan-microglial marker, ionised calcium-binding adapter molecule 1 (IBA1), Aβ and tau, was performed in the primary motor cortex (PMC), a region relatively spared of AD pathological changes, and compared to the severely affected inferior temporal cortex (ITC) in the same cases. Unlike the ITC, the PMC in the AD cases was spared of any degenerative changes in cortical thickness and the density of Betz cells and total neurons. The clustering of activated microglia was greatest in the PMC of AD cases and high pathology controls compared to the ITC. This suggests microglial activation is most prominent in the early phases of AD pathophysiology. Nascent tau inclusions were found in neuritic plaques in the PMC but were more numerous in the ITC of the same case. This shows that tau positive neuritic plaques begin early in AD which is likely of pathogenic importance, however major tau deposition follows the accumulation of Aβ and clustering of activated microglia. Importantly, findings presented here demonstrate that different states of microglial activation, corresponding to regional accumulations of Aβ and tau, are present simultaneously in the same individual; an important factor for consideration if targeting these cells for therapeutic intervention.
Arathi Jayaraman, Karen Avgush, Rashad Kulam, Advait Soni, Areeb Khan, Mourad Kerdjoudj, Sundararajan Jayaraman
Free Neuropathol 1: 19 (2020)
We have previously shown that treatment of female NOD mice with a potent nonselective histone deacetylase inhibitor attenuated experimental autoimmune encephalomyelitis, a model for progressive multiple sclerosis. Herein we show that immunization with the MOG35-55 peptide induced prolonged upregulation of genes encoding interleukin 17A (IL-17A), aryl hydrocarbon receptor, and histone deacetylase 11 in the spinal cord whereas the subunits of IL-27, IL-27p28 and IL-27ebi3 were significantly increased in secondary lymphoid organs after a lag period. Interestingly, the nitric oxide synthase gene was prominently expressed in both of these anatomic compartments following immunization. Treatment with the histone modifier repressed the transcription of all of these genes induced by immunization. Moreover, the drug suppressed the steady-state levels of the migration inhibitory factor and CD274 genes in both the spinal cord and peripheral lymphoid tissues. At the same time, the CD39 gene was downregulated only in secondary lymphoid organs. Paradoxically, the epigenetic drug enhanced the expression of Declin-1 in the spinal cord, suggesting a protective role in neuronal disease. Immunization profoundly enhanced transcription of the chemokine CCL2 in the secondary lymphoid tissues without a corresponding increase in the translation of CCL2 protein. Histone hyperacetylation neither altered the transcription of CCL2 nor its cognate receptor CCR2 in the central nervous system and peripheral lymphoid tissues. Surprisingly, the drug did not exert modulatory influence on most other immune response-related genes previously implicated in encephalomyelitis. Nevertheless, our data uncover several potential molecular targets for the intervention of experimental autoimmune encephalomyelitis that have implications for the treatment of progressive multiple sclerosis.
Kevin F Bieniek, Keith Anthony Josephs, Wen-Lang Lin, Dennis W Dickson
Free Neuropathol 1: 9 (2020)
Background: The majority of cases of frontotemporal lobar degeneration (FTLD) are characterized by focal cortical atrophy with an underlying tau or TDP-43 proteinopathy. A subset of FTLD cases, however, lack tau and TDP-43 immunoreactivity, but have neuronal inclusions positive for ubiquitin, referred to as atypical FTLD (aFTLD-U). Studies have demonstrated that ubiquitin-positive inclusions in aFTLD-U are immunoreactive for fused in sarcoma (FUS). As such, the current nosology for this entity is FTLD-FUS, which is thought to include not only aFTLD-U but also neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease.

Objective: To compare pathological features of cases of aFTLD-U and NIFID.

Methods: We reviewed the neuropathology of 15 patients (10 males and 5 females; average age at death 54 years (range 41-69 years)) with an antemortem clinical diagnosis of a frontotemporal dementia and pathological diagnosis of aFTLD-U (n=8) or NIFID (n=7). Sections were processed for immunohistochemistry and immunoelectron microscopy with FUS, TDP-43, and α-internexin (αINX) antibodies.

Results: Eight cases had pathologic features consistent with FTLD-FUS, with severe striatal atrophy (7/8 cases), as well as FUS-positive neuronal cytoplasmic and vermiform intranuclear inclusions, but no αINX immunoreactivity. Five cases had features consistent with NIFID, with neuronal inclusions positive for both FUS and αINX. Striatal atrophy was present in only two of the NIFID cases. Two cases had αINX-positive neuronal inclusions consistent with NIFID, but both lacked striatal atrophy and FUS immunoreactivity. Surprisingly, one of these two NIFID cases had lesions immunoreactive for TDP-43.

Discussion: While FUS pathology remains a prominent feature of aFTLD-U, there is pathologic heterogeneity, including rare cases of NIFID with TDP-43- rather than FUS-positive inclusions.
Ellen Gelpi, Naomi P. Visanji, Selma Hönigschnabl, Angelika Reiner, Peter Fischer, Anthony Lang, Herbert Budka, Gabor G. Kovacs
Free Neuropathol 1: 6 (2020)
Deposition of alpha-synuclein in the brain is a hallmark of Lewy body disorders. Alpha-synuclein has been considered to show prion-like properties. Prion diseases can be transmitted by the transplantation of cadaveric dura mater causing iatrogenic Creutzfeldt-Jakob disease. Recent observations of amyloid-β deposition in dural grafts support the seeding properties of amyloid-β. Here we assessed the presence of alpha-synuclein in dura mater samples as a potential transmissible seed source. We immunostained 32 postmortem dura mater samples; 16 cases with Lewy-body disorder (LBD) showing different pathology stages and 16 non-LBD cases for phosphorylated (Ser129) and disease-associated (5G4) alpha-synuclein. Disease-associated alpha-synuclein aggregates were identified in intradural nerve fibres and associated with a vessel in a single LBD-Braak stage 4 case. We conclude that alpha-synuclein is detectable, although rarely, in dura mater samples in patients with LBD. The risk of potential transmissibility of dural alpha-synuclein deserves assessment by complementary experimental studies.