Neuropathology and Applied Neurobiology (2023)

Muscleblind Knockout 2 alters the expression of the aductin-1 isoform and alters the dynamics of the dendritic column of cortical neurons during brain development

Turning off the blind muscle type 2 gene alters the expression of the aductin-1 isoform and alters the dynamics of the dendritic column in cortical neurons during brain development

Muscleblind-like 2 (MBNL2) plays a critical role in regulating alternative splicing during development, and loss of MBNL2 in mice recapitulates brain phenotypes in myotonic dystrophy (DM). However, the mechanisms underlying the neuropathogenesis of DM during brain development remain unclear. In this study, we aim to investigate the impact of MBNL2 knockout on neuronal development using Mbnl2 conditional knockout (CKO) mouse models. To generate Mbnl2 knockout neurons, cDNA encoding Cre recombinase was electroporated in utero into neuronal progenitor cells from brains of Mbnl2flox/flox mice. Dendritic spine morphology and dynamics were monitored by confocal and two-photon microscopy in brain slices and live animals from neonatal to adulthood. To investigate the underlying molecular mechanism, we also identified changes in splicing and molecular interactions of proteins associated with spinogenesis. MBNL2 plays a critical role in maintaining dendritic spine dynamics and homeostasis in the developing brain. Mis-splicing of ADD1 downstream could be responsible for the changes and contribute to the cerebral pathogenesis of DM.

Muscle blind-type protein 2 (MBNL2) plays a crucial role in the regulation of alternative splicing during development, and deletion of MBNL2 in mice recapitulates the brain phenotype of myotonic dystrophy (DM). However, the neuropathogenesis of DM during brain development is still unclear. In this study, we aimed to investigate the effects of MBNL2 ablation on neuronal development using the MbnL2 conditional knockout (CKO) mouse model. To establish Mbnl2 knockout neurons, the gene encoding Cre recombinase was introduced into neuronal progenitor cells in the brain of Mbnl2-flox/flox mice by electroporation in utero. The morphology and dynamics of dendritic spines in brain slices and in live animals from neonatal to adulthood were observed using confocal microscopy and two-photon microscopy. To probe the underlying molecular mechanism, we uncovered additional changes in the splicing and molecular interactions of proteins associated with spinal development. MBNL2 plays a key role in maintaining dendritic spine homeostasis and homeostasis in the developing brain. Downstream mis-splicing of ADD1 could be responsible for these changes and be involved in the pathogenesis of brain DM.

REF: Huang CW, Lee KY, Lin PT, et al. Muscleblind-like 2 knockout alters the expression of the aductin-1 isoform and alters the dynamics of the dendritic column of cortical neurons during brain development. Neuropathol Appl Neurobiol. 2023;49(2):e12890. doi:10.1111/nan.12890 PMID: 36765387

Effects of mutant huntingtin on oxytocin neurons in non-motor features of HD

Effects of mutant huntingtin on oxytocin neurons in HD patients on non-motor function

Early non-motor characteristics are present in Huntington's disease (HD), including anxiety, depression and altered social perceptions. The underlying neurobiological mechanisms are not known. Oxytocin (OXT) is involved in the regulation of emotions, social cognition and metabolism, and our previous work has shown that the OXT system is affected early in HD. The aim of the study was to investigate the possible causal relationship between the selective expression of mutant huntingtin (mHTT) in OXT neurons and the development of non-motor traits and neuropathology. To express mHTT only in OXT neurons, we used a novel flex-switch adeno-associated viral vector design to selectively express mHTT or wild-type HTT in the paraventricular nucleus of the hypothalamus using OXT-Cre recombinase mice. We also performed a mirror experiment to selectively exclude mHTT in OXT neurons using the BACHD mouse model. Mice underwent a battery of behavioral tests three months after injection or at two months of age to assess their psychiatric and social behavior. Post-mortem analyzes were performed to assess the impact on the OXT system. This study demonstrates that mHTT expression can exert autonomic toxic effects on OXT neuron cells without affecting the non-motor phenotype at early time points in mice.

Early non-motor characteristics of Huntington's disease (HD) include anxiety, depression and social cognitive impairment. The underlying neurobiological mechanism is still unclear. Oxytocin (OXT) is involved in the regulation of emotions, social cognition and metabolism, and our previous work has shown that the OXT system is impaired in early HD. The aim of this study was to investigate the possible causal relationship between the selective expression of mutant huntingtin (MHTT) in OXT neurons and the development of non-motor functions and neuropathology. To express mHTT only in oxt neurons, we use a novel flex-switch adeno-associated virus vector design using mouse oxt-Cre recombinase to selectively express mHTT or wild-type HTT in the paraventricular nucleus of the hypothalamus. We also performed mirror experiments using the BACHD mouse model to selectively delete mHTT in OXT neurons. Mice underwent a battery of behavioral tests at three and two months of age after injection to assess their psychosocial and social behavior. An autopsy analysis was performed to assess the impact on the OXT System. This study demonstrates that mHTT expression can induce cell-autonomous toxic effects in OXT neurons without affecting the early non-motor phenotype of mice.

REF: Bergh S, Gabery S, Tonetto S, Kirik D, Petersén Å, Cheong RY. Effects of mutant huntingtin on oxytocin neurons in non-motor features of HD. Neuropathol Appl Neurobiol. 2023;49(2):e12891. doi:10.1111/nan.12891 PMID: 36776123

A standardized protocol for collecting and processing blood and CSF for biomarker research in ataxia

Standardized protocols for collecting and processing blood and CSF for ataxia biomarker studies

The European Spinocerebellar Ataxia Type 3/Machado-Joseph Disease Initiative (ESMI) is a consortium aiming to establish the largest European cohort suitable for longitudinal studies of Spinocerebellar Ataxia Type 3/Machado-Joseph Disease (SCA3/MJD), the ataxia , is the world's most common autosomal dominant disease. One of ESMI's main focus areas was the identification of SCA3/MJD biomarkers to enable future interventional studies. Because biosampling collection and processing variables have a significant impact on the results of biomarker studies, standardization of biosampling procedures was performed prior to the start of the study visit. Here we describe the ESMI consensus biosampling protocol that was developed within ESMI and can ultimately be transferred to other neurodegenerative diseases, particularly ataxias, representing the first step towards protocol harmonization in this field.

The European Spinocerebellar Ataxia 3/Machado-Joseph Disease Initiative (ESMI) is an initiative that aims to facilitate the largest longitudinal study of Spinocerebellar Ataxia 3/Machado-Joseph Disease (SCA3/MJD) in the European Alliance of Ready Cohorts, SCA3/MJD, to establish the world's most common autosomal dominant ataxia. One of ESMI's main focus areas is the identification of SCA3/MJD biomarkers to enable future interventional studies. Because the variables of biosampling and processing have a significant impact on the results of biomarker studies, previous standardization of biosampling procedures began with study visits. Here we describe the ESMI Consensus Biosampling Protocol, which was developed in the context of ESMI and could potentially be transferred to other neurodegenerative diseases, particularly ataxia, as a first step towards harmonizing protocols in this area.

REF: Santana MM, Gaspar LS, Pinto MM, et al. A standardized protocol for collecting and processing blood and CSF for biomarker research in ataxia. Neuropathol Appl Neurobiol. 2023;49(2):e12892. doi:10.1111/nan.12892 PMID: 36798010

Pathways to hippocampal damage in neuromyelitis optica spectrum disorders

Hippocampal damage pathways in neuromyelitis optica spectrum disorders

Many patients with neuromyelitis optica spectrum disorders (NMOSD) have cognitive impairments that affect memory, processing speed, and attention, and experience depressive symptoms. Because some of these manifestations are hippocampal, several magnetic resonance imaging (MRI) studies have been performed in the past, with several groups describing hippocampal volume loss in patients with NMOSD, while others did not observe such changes. Here we address these discrepancies. We performed pathologic and MRI studies on the hippocampus of patients with NMOSD combined with detailed immunohistochemical analysis of the hippocampus using experimental NMOSD models. Various pathologic scenarios can result in hippocampal volume loss in patients with NMOSD.

Many people with neuromyelitis optica spectrum disorder (NMOSD) have cognitive impairments that affect memory, processing speed, and concentration, as well as depressive symptoms. Because some of these manifestations can be attributed to the hippocampus, several magnetic resonance imaging (MRI) studies have been performed in the past, with some groups describing reduced hippocampal volume in patients with NMOSD, while others did not observe such changes. Here we address these differences. We performed pathologic and MRI studies of the hippocampus of patients with NMOSD and detailed immunohistochemical analysis of the hippocampus of an experimental NMOSD model. Various pathologic conditions can result in decreased hippocampal volume in patients with NMOSD.

REF: Zakani M, Nigritinou M, Ponleitner M, et al. Pathways to hippocampal damage in neuromyelitis optica spectrum disorders. Neuropathol Appl Neurobiol. 2023;49(2):e12893. doi:10.1111/nan.12893 PMID: 36811295

Classification of glial neuronal tumors based on DNA methylation works in concert with histology and radiology to refine precise molecular stratification

Synergy of DNA methylation-based glioma classification with histology and radiology to refine precise molecular layering

Glioneuronal tumors (GNTs) are poorly characterized by their histology and have no reliable diagnostic indicators. Previously, we showed that common GNTs consist of two molecularly distinct groups that correlate poorly with histology. To refine the diagnosis, we created a methylation-based model for GNT classification and further evaluated the molecular stratification patterns using methylation, histology, and radiology. We extensively analyzed methylation, radiology and histology for 83 GNT samples: a training cohort of 49 previously classified into molecularly defined groups based on genomic profiles, plus a validation cohort of 34. We identified histological and radiological correlates for molecular classification and generated a basis on the Support Vector Machine (SVM) model for forecasting. We then compared methylation and radiological and histological classifications in validation NTGs. The diagnostic criteria reflect the biology of the glioneuronal tumor only insufficiently, so that an indissoluble part remains. In the largest cohort of molecularly defined glioneuronal tumors, we developed molecular, histological, and radiological approaches for biologically meaningful classification and showed that almost all cases are solvable, underscoring the importance of an integrated diagnostic approach.

Glioneurocytic tumors (GNTs) are poorly differentiated histologically and have no reliable diagnostic markers. Previously, we found that common GNTs consisted of two molecularly distinct groups that correlated poorly with histology. To refine the diagnosis, we created a methylation-based GNT classification model and then evaluated molecular stratification criteria for methylation, histology, and radiology. We extensively analyzed methylation, radiology, and histology in 83 GNT samples: 49 training cohorts, previously classified into molecularly defined groups based on genomic profiles, plus 34 validation cohorts. We identified histological and radiological correlations with molecular classification and constructed methylation-based Support Vector Machine (SVM) models for prediction. We then compared the role of methylation and radiological and histological classification in the validation of the GNTS. The diagnostic criteria do not adequately reflect the biology of glioma, leaving a proportion unexplained. In the largest cohort of molecularly defined gliomas, we developed molecular, histological, and radiological approaches for biologically meaningful classification and showed that almost all cases resolved, highlighting the importance of an integrated diagnostic approach.

REF: Stone TJ, Mankad K, Tan AP, et al. Classification based on DNA methylation of glial neuron tumors works in concert with histology and radiology to refine precise molecular stratification. Neuropathol Appl Neurobiol. 2023;49(2):e12894. doi:10.1111/nan.12894 PMID: 36843390

Schwann cells and myelin in human peripheral nerve: The major protein components vary with age, axon size, and pathology

Schwann cells and myelin in human peripheral nerves: changes in major protein components with age, axon size, and pathological changes

We examined the major protein components of Schwann cells (SCs) and myelin in normal and diseased human peripheral nerves. We examined the distributions of neural cell adhesion molecule (NCAM), P0 protein (P0), and myelin basic protein (MBP) in frozen sections of 98 sural nerves. Peripheral nerve FB and myelin exhibit distinct molecular phenotypes related to age, axon size, and nerve pathology. In the normal adult peripheral nerve, myelin exhibits two distinct patterns of molecular composition. MBP is virtually absent in the myelin surrounding a population of medium-sized axons, whereas P0 is present in the myelin surrounding all axons. Denervated SCs have a molecular signature that differs from normal SC types. In acute denervation, SCs can stain both NCAM and MBP. Chronically denervated SCs usually stain on NCAM and P0.

We examined the major protein components of Schwann cells (SCs) and myelin in normal and diseased human peripheral nerves. In this work, the distribution of nerve cell adhesion molecule (NCAM), P0 protein (P0) and myelin basic protein (MBP) were observed in frozen sections of 98 sural nerves. Peripheral neural stem cells and myelin exhibit distinct molecular phenotypes related to age, axon size, and neuropathology. In normal adult human peripheral nerves, myelin exhibits two distinct patterns of molecular composition. MBP is largely absent in the myelin sheath around a population of medium-sized axons, while P0 is present in the myelin sheath around all axons. Denervated stem cells exhibit molecular properties that differ from those of normal stem cell types. In the acute denervated state, stem cells can stain NCAM and MBP. Chronically denervated SCs normally express NCAM and P0.

REF: Pestronk A, Schmidt RE, Bucelli R, Sim J. Schwann cells and myelin in human peripheral nerve: major protein components vary with age, axon size, and pathology. Neuropathol Appl Neurobiol. 2023;49(2):e12898. doi:10.1111/nan.12898 PMID: 36868780

Patient-derived xenograft mouse models to study the central nervous system and retinal tropism of primary and secondary central nervous system lymphomas

Patient-derived xenograft mouse model to study the tropism of primary and secondary lymphomas from the CNS to the CNS and to the retina

How and why lymphoma cells lodge in the central nervous system and in the vitreoretinal compartment in primary diffuse large B-cell lymphoma of the central nervous system remains unknown. Our aim was to create an in vivo model to study the tropism of lymphoma cells towards the central nervous system. We constructed a patient-derived xenograft mouse model of central nervous system lymphoma and characterized xenografts from four patients with primary and four secondary central nervous system lymphomas using immunohistochemistry, flow cytometry, and nucleic acid sequencing technology. In reimplantation experiments, we analyzed propagation patterns of orthotopic and heterotopic xenografts and performed RNA sequencing of different involved organs to detect differences at the transcriptome level. Our in vivo tumor model preserves key features of primary and secondary central nervous system lymphoma and can be used to explore critical pathways to the central nervous system and retinal tropism with the aim of finding new targets for new therapeutic approaches.

It remains unclear how and why lymphoma cells colonize the CNS and vitreoretinal septum in primary diffuse CNS large B-cell lymphoma. Our aim was to develop an in vivo model to study the tropism of lymphoma cells towards the CNS. We constructed a patient-derived xenograft model for central nervous system lymphoma and analyzed xenografts from 4 patients with primary and 4 secondary central nervous system lymphomas using immunohistochemistry, flow cytometry, and nucleic acid sequencing. In retransplantation experiments, we analyzed diffusion patterns of orthotopic and heterotopic xenografts and performed RNA sequencing of different affected organs to detect differences at the transcriptome level. Our in vivo tumor model retains key features of primary and secondary CNS lymphomas and can be used to explore important signaling pathways in the CNS and retinal tropism with the aim of finding new targets for new therapeutic approaches.

REF: Isbell LK, Tschuch C, Doostkam S, et al. Patient-derived xenograft mouse models to study the central nervous system and retinal tropism of primary and secondary central nervous system lymphomas. Neuropathol Appl Neurobiol. 2023;49(2):e12899. doi:10.1111/nan.12899 PMID: 36879456

Occurrence of focal myositis during Behçet's disease: identification of a specific focal myopathy associated with vasculitis

Occurrence of focal myositis during Behçet's disease: identification of a specific focal myopathy associated with vasculitis

The aim of this study was to demonstrate the association between focal myositis (FM) and Behçet's disease (BD) and to analyze the main features of this association. This is a retrospective multicenter study of patients with BD and FM (BD + FM+ group) and without FM (BD - FM+ group). Clinical, laboratory, radiological, pathological, treatment and outcome data were analyzed. The FM that occurs during BD appears to be part of the systemic vasculitis process and presents as a focal myopathy associated with vasculitis with a specific clinical-histological pattern. Patients with this association require long-term follow-up and appropriate treatment. This case series also highlights the need for research on diagnostic criteria for BD in FM cases.

The aim of this study is to demonstrate the association between focal myositis (FM) and Behçet's disease (BD) and to analyze the main features of this association. This is a multicenter retrospective study of patients with BD combined with FM (BD+FM+ group) and non-FM (BD-FM+ group). Clinical, laboratory, radiological, pathological, treatment and outcome data were analyzed. The FM that occurs during BD appears to be part of a systemic vasculitic process that manifests as focal myopathy associated with vasculitis with a specific histological clinical pattern. Patients with this association require long-term follow-up and appropriate treatment. This case series also highlights the need for research on diagnostic criteria for BD in FM cases.

REF: Gallay L, Hot A, Allenbach Y, et al. Occurrence of focal myositis during Behçet's disease: identification of a specific focal myopathy associated with vasculitis. Neuropathol Appl Neurobiol. 2023;49(2):e12900. doi:10.1111/nan.12900PMID: 36919233

TDP-43 pathology and functional deficits in wild-type cyclin F mutant and ALS/FTD mouse models

Pathologic and functional defects of TDP-43 in wild-type and ALS/FTD mutant cyclin F mouse models

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons, leading to muscle weakness and eventual death. Frontotemporal dementia (FTD) manifests itself clinically in a significant loss of behavior. About 10% of cases have a known family history, and disease-related mutations in multiple genes have been identified in FTD and ALS. More recently, ALS- and FTD-associated variants have been identified in the CCNF gene, which accounts for approximately 0.6% to over 3% of familial ALS cases. In this study, we developed the first mouse models expressing wild-type human CCNF (WT) or its mutant pathogenic variant S621G to summarize the main clinical and neuropathological features of ALS and FTD associated with CCNF disease variants. We express human CCNF WT or CCNFS621G in whole mouse brain by intracranial delivery of adeno-associated virus (AAV) to achieve widespread delivery by somatic brain transgenesis. In conclusion, CCNF expression in mice reproduces the clinical presentation of ALS, including functional deficits and TDP-43 neuropathology, with altered CCNF-mediated signaling pathways contributing to the observed pathology.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of upper and lower motor neurons, leading to muscle weakness and eventual death. Frontotemporal dementia (FTD) manifests itself clinically in a significant behavioral decline. About 10 percent of cases have a known family history, and multiple disease-associated gene mutations have been found in FTD and ALS. Recently, ALS- and FTD-associated variants in the Ccnf gene have been identified, which are estimated to account for 0.6% to over 3% of familial ALS cases. In this study, we established the first mouse model expressing wild-type (WT) human Ccnf or its mutant pathogenic variant S621G to summarize the main clinical and neurological disorders of ALS and FTD related to the physical features of pathogenic Ccnf variants. We express human Ccnf WT or CCNFS621G in mouse brains by intracerebral delivery of adeno-associated virus (AAV), thus achieving broad delivery through somatic brain transgenesis. In conclusion, Ccnf expression in mice summarizes the clinical manifestations of ALS, including functional deficits and TDP-43 neuropathology, with Ccnf-mediated signaling pathways altering the observed pathology.

REF: van Hummel A, Sabale M, Przybyla M, et al. TDP-43 pathology and functional deficits in selvage-type mutant cyclin F walker models and ALS/FTD. Neuropathol Appl Neurobiol. 2023;49(2):e12902. doi:10.1111/nan.12902 PMID: 36951214