Clinical and neuroanatomical characterization of the semantic behavioral variant of frontotemporal dementia in a multicenter Italian cohort.

BACKGROUND: Semantic behavioral variant frontotemporal dementia (sbvFTD) is a neurodegenerative condition presenting with specific behavioral and semantic derangements and predominant atrophy of the right anterior temporal lobe (ATL). The objective was to evaluate clinical, neuropsychological, neuroimaging, and genetic features of an Italian sbvFTD cohort, defined according to recently proposed guidelines, compared to semantic variant primary progressive aphasia (svPPA) and behavioral variant FTD (bvFTD) patients. METHODS: Fifteen sbvFTD, sixty-three bvFTD, and twenty-five svPPA patients and forty controls were enrolled. Patients underwent clinical, cognitive evaluations, and brain MRI. Symptoms of bvFTD patients between onset and first visit were retrospectively recorded and classified as early and late. Grey matter atrophy was investigated using voxel-based morphometry. RESULTS: sbvFTD experienced early criteria-specific symptoms: world, object and person-specific semantic loss (67%), complex compulsions and rigid thought (60%). Sequentially, more behavioral symptoms emerged (apathy/inertia, loss of empathy) along with non-criteria-specific symptoms (anxiety, suspiciousness). sbvFTD showed sparing of attentive/executive functions, especially compared to bvFTD and better language functions compared to svPPA. All sbvFTD patients failed at the famous face recognition test and more than 80% failed in understanding written metaphors and humor. At MRI, sbvFTD had predominant right ATL atrophy, almost specular to svPPA. Three sbvFTD patients presented pathogenic genetic variants. CONCLUSION: We replicated the application of sbvFTD diagnostic guidelines in an independent Italian cohort, demonstrating that the presence of person-specific semantic knowledge loss and mental rigidity, along with preserved executive functions and a predominant right ATL atrophy with sparing of frontal lobes, should prompt a diagnosis of sbvFTD.

Dissecting the Clinical Heterogeneity and Genotype-Phenotype Correlations of MAPT Mutations: A Systematic Review.

BACKGROUND: Microtubule-associated protein tau (MAPT) mutations are one of the main causes of genetic Frontotemporal dementia (FTD) and are characterised by high clinical heterogeneity. A behavioural variant of FTD is the principal phenotype, but other rarer phenotypes are described, mostly reported as single cases. In this review, we provide an overview of the clinical phenotypes associated with MAPT mutations in order to define their characteristics and explore genotype-phenotype correlations. METHODS: We performed systematic bibliographic research on the Pubmed database, focusing on articles published between 1998 and 2022. We analysed the clinical phenotype of 177 patients carrying MAPT mutations, focusing on the rarest ones. We performed a narrative synthesis of the results. RESULTS: Regarding language phenotypes, the most frequent were the non-fluent variant and the semantic variant of Primary Progressive Aphasia (nfvPPA, svPPA), approximately in the same proportion. Almost 20% of the whole group of patients present a clinical phenotype belonging to the corticobasal syndrome-progressive supranuclear palsy (CBS-PSP) spectrum. While no clear genotype-phenotype correlation could be identified, some mutations were associated with a specific phenotype, while others gave origin to multiple clinical pictures and mixed phenotypes. CONCLUSIONS: A high clinical heterogeneity exists in FTD associated with MAPT mutations without a clear phenotype-genotype correlation in most cases. However, some characteristics can be helpful to drive genetic testing. Deep phenotyping of patients, together with functional studies of single mutations, particularly those associated with atypical phenotypes, are necessary to better understand the biological mechanisms underlying this clinical variability.

Functional Connectivity From Disease Epicenters in Frontotemporal Dementia.

BACKGROUND AND OBJECTIVES: MRI connectomics is an ideal tool to test a network-based model of pathologic propagation from a disease epicenter in neurodegenerative disorders. In this study, we used a novel graph theory-based MRI paradigm to explore functional connectivity reorganization, discerning between direct and indirect connections from disease epicenters, and its relationship with neurodegeneration across clinical presentations of the frontotemporal dementia (FTD) spectrum, including behavioral variant of FTD (bvFTD), nonfluent variant of primary progressive aphasia (nfvPPA), and semantic variant of primary progressive aphasia (svPPA). METHODS: In this observational cross-sectional study, disease epicenters were defined as the peaks of atrophy of a cohort of patients with high confidence of frontotemporal lobar degeneration pathology (Mayo Clinic). These were used as seed regions for stepwise functional connectivity (SFC) analyses in an independent (Milan) set of patients with FTD to assess connectivity in regions directly and indirectly connected to the epicenters. Correlations between SFC architecture in healthy conditions and atrophy patterns in patients with FTD were also tested. RESULTS: As defined by comparing the 42 Mayo Clinic patients with 15 controls, disease epicenters were the left anterior insula for bvFTD, left supplementary motor area for nfvPPA, and left inferior temporal gyrus (ITG) for svPPA. Compared with 94 age-matched controls, patients with bvFTD (n = 64) and nfvPPA (n = 34) of the Milan cohort showed widespread decreased SFC in bilateral cortical regions with direct/indirect connections with epicenters and increased SFC either in directly connected regions, physically close to the respective seed region, or in more distant cortical/cerebellar areas with indirect connections. Across all link steps, svPPA (n = 36) showed SFC decrease mostly within the temporal lobes, with co-occurrent SFC increase in cerebellar regions at indirect link steps. The average stepwise topological distance from the left ITG in a reference group of 50 young healthy controls correlated with regional gray matter volume in svPPA, consistent with network-based degeneration. DISCUSSION: Our findings demonstrate that each FTD syndrome is associated with a characteristic interplay of decreased and increased functional connectivity with the disease epicenter, affecting both direct and indirect connections. SFC revealed novel insights regarding the topology of functional disconnection across FTD syndromes, holding the promise to be used to model disease progression in future longitudinal studies.

24-Hydroxycholesterol Induces Tau Proteasome-Dependent Degradation via the SIRT1/PGC1α/Nrf2 Pathway: A Potential Mechanism to Counteract Alzheimer's Disease.

Considerable evidence indicates that cholesterol oxidation products, named oxysterols, play a key role in several events involved in Alzheimer's disease (AD) pathogenesis. Although the majority of oxysterols causes neuron dysfunction and degeneration, 24-hydroxycholesterol (24-OHC) has recently been thought to be neuroprotective also. The present study aimed at supporting this concept by exploring, in SK-N-BE neuroblastoma cells, whether 24-OHC affected the neuroprotective SIRT1/PGC1α/Nrf2 axis. We demonstrated that 24-OHC, through the up-regulation of the deacetylase SIRT1, was able to increase both PGC1α and Nrf2 expression and protein levels, as well as Nrf2 nuclear translocation. By acting on this neuroprotective pathway, 24-OHC favors tau protein clearance by triggering tau ubiquitination and subsequently its degradation through the ubiquitin-proteasome system. We also observed a modulation of SIRT1, PGC1α, and Nrf2 expression and synthesis in the brain of AD patients with the progression of the disease, suggesting their potential role in neuroprotection. These findings suggest that 24-OHC contributes to tau degradation through the up-regulation of the SIRT1/PGC1α/Nrf2 axis. Overall, the evidence points out the importance of avoiding 24-OHC loss, which can occur in the AD brain, and of limiting SIRT1, PGC1α, and Nrf2 deregulation in order to prevent the neurotoxic accumulation of hyperphosphorylated tau and counteract neurodegeneration.

Corrigendum: PMCA-based detection of prions in the olfactory mucosa of patients with sporadic Creutzfeldt-Jakob disease.

[This corrects the article DOI: 10.3389/fnagi.2022.848991.].

Semantic and right temporal variant of FTD: Next generation sequencing genetic analysis on a single-center cohort.

Semantic and right temporal variant of frontotemporal dementia (svFTD and rtvFTD) are rare clinical phenotypes in which, in most cases, the underlying pathology is TDP-43 proteinopathy. They are usually sporadic disorders, but recent evidences suggest a higher frequency of genetic mutations for the right temporal versus the semantic variant. However, the genetic basis of these forms is not clear. In this study we performed a genetic screening of a single-center cohort of svFTD and rtvFTD patients, aiming at identifying the associated genetic variants. A panel of 73 dementia candidate genes has been analyzed by NGS target sequencing including both causal and risk/modifier genes in 23 patients (15 svFTD and 8 rtvFTD) and 73 healthy age-matched controls. We first performed a single variant analysis considering rare variants and then a gene-based aggregation analysis to evaluate the cumulative effects of multiple rare variants in a single gene. We found 12 variants in nearly 40% of patients (9/23), described as pathogenic or classified as VUS/likely pathogenic. The overall rate was higher in svFTD than in rtvFTD. Three mutations were located in MAPT gene and single mutations in the following genes: SQSTM1, VCP, PSEN1, TBK1, OPTN, CHCHD10, PRKN, DCTN1. Our study revealed the presence of variants in genes involved in pathways relevant for the pathology, especially autophagy and inflammation. We suggest that molecular analysis should be performed in all svFTD and rtvFTD patients, to better understand the genotype-phenotype correlation and the pathogenetic mechanisms that could drive the clinical phenotypes in FTD.

Significance and clinical suggestions for the somatosensory evoked potentials increased in amplitude revealed by a large sample of neurological patients.

OBJECTIVES: To investigate the relationship between N20-P25 peak-to-peak amplitude (N20p-P25p) of somatosensory evoked potentials (SEPs) and the occurrence of abnormalities of the peripheral and/or central sensory pathways and of myoclonus/epilepsy, in 308 patients with increased SEPs amplitude from upper limb stimulation. METHODS: We compared cortical response (N20p-P25p) in different groups of patients identified by demographic, clinical, and neurophysiological factors and performed a cluster analysis for classifying the natural occurrence of subgroups of patients. RESULTS: No significant differences of N20p-P25p were found among different age-dependent groups, and in patients with or without PNS/CNS abnormalities of sensory pathways, while myoclonic/epileptic patients showed higher N20p-P25p than other groups. Cluster analysis identified four clusters of patients including myoclonus/epilepsy, central sensory abnormalities, peripheral sensory abnormalities, and absence of myoclonus and sensory abnormalities. CONCLUSIONS: Increased N20p-P25p prompts different possible pathophysiological substrates: larger N20p-P25p in patients with cortical myoclonus and/or epilepsy is likely sustained by strong cortical hyperexcitability, while milder increase of N20p-P25p could be underpinned by plastic cortical changes following abnormalities of sensory pathways, or degenerative process involving the cortex. SEPs increased in amplitude cannot be considered an exclusive hallmark of myoclonus/epilepsy. Indeed, in several neurological disorders, it may represent a sign of adaptive, plastic, and/or degenerative cortical changes.

Neurosyphilis Mimicking Behavioral Variant of Frontotemporal Dementia in a 59-Year-Old Man.

We present the case of a man exhibiting a clinical phenotype of behavioral variant of frontotemporal dementia (bvFTD). The man had developed psychiatric disturbances with verbal aggressiveness over a few months, followed by cognitive and frontal behavioral disorders, fulfilling the clinical criteria for bvFTD. Atrophy and hypometabolism in frontotemporal regions were consistent with the diagnosis. However, serum-screening exams for syphilis infection were positive, and CSF analysis, despite a negative Venereal Disease Research Laboratory Test, suggested the diagnosis of neurosyphilis. After specific antibiotic therapy, the man's behavioral abnormalities and cognitive deficits notably improved, confirming neurosyphilis as the cause of the clinical phenotype. The cognitive deficits completely recovered 1 year post therapy and remained stable for 2 years. After ∼2½ years from the first treatment, the man's behavioral disorders mildly worsened, at which time we re-evaluated him. His cognition was stable, and a positive Venereal Disease Research Laboratory Test confirmed the diagnosis of neurosyphilis. With this case, we demonstrated that in some instances, neurosyphilis can mimic frontotemporal dementia. As a cause of treatable dementia, it should be considered in the differential diagnosis of bvFTD, particularly when psychiatric symptoms and a rapid cognitive decline are noted, even in the presence of brain atrophy and/or hypometabolism.

Resting state functional brain networks associated with emotion processing in frontotemporal lobar degeneration.

This study investigated the relationship between emotion processing and resting-state functional connectivity (rs-FC) of the brain networks in frontotemporal lobar degeneration (FTLD). Eighty FTLD patients (including cases with behavioral variant of frontotemporal dementia, primary progressive aphasia, progressive supranuclear palsy syndrome, motor neuron disease) and 65 healthy controls underwent rs-functional MRI. Emotion processing was tested using the Comprehensive Affect Testing System (CATS). In patients and controls, correlations were investigated between each emotion construct and rs-FC changes within critical networks. Mean rs-FC of the clusters significantly associated with CATS scoring were compared among FTLD groups. FTLD patients had pathological CATS scores compared with controls. In controls, increased rs-FC of the cerebellar and visuo-associative networks correlated with better scores in emotion-matching and discrimination tasks, respectively; while decreased rs-FC of the visuo-spatial network was related with better performance in the affect-matching and naming. In FTLD, the associations between rs-FC and CATS scores involved more brain regions, such as orbitofrontal and middle frontal gyri within anterior networks (i.e., salience and default-mode), parietal and somatosensory regions within visuo-spatial and sensorimotor networks, caudate and thalamus within basal-ganglia network. Rs-FC changes associated with CATS were similar among all FTLD groups. In FTLD compared to controls, the pattern of rs-FC associated with emotional processing involves a larger number of brain regions, likely due to functional specificity loss and compensatory attempts. These associations were similar across all FTLD groups, suggesting a common physiopathological mechanism of emotion processing breakdown, regardless the clinical presentation and pattern of atrophy.

PMCA-Based Detection of Prions in the Olfactory Mucosa of Patients With Sporadic Creutzfeldt-Jakob Disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials.

MAPT Q336H mutation: Intrafamilial phenotypic heterogeneity in a new Italian family.

BACKGROUND AND PURPOSE: Q336H is a rare MAPT mutation, previously found in a single patient with behavioral variant frontotemporal dementia and tau pathology (Pick bodies). Here, we describe the clinical characteristics of two members of a new family carrying the Q336H MAPT mutation. METHODS: Clinical, genetic, and neuroradiological assessment and follow-up of the proband were made. RESULTS: At age 37 years, the proband developed naming and object recognition impairment, due to a lack of knowledge. After 3 years, he developed behavioral disorders. Magnetic resonance imaging (MRI) and fluorodeoxyglucose positron emission tomography showed the involvement of the left temporal pole. A diagnosis of semantic variant primary progressive aphasia (svPPA) was made. At follow-up after 6 and 12 months, a rapid worsening of cognitive deficits occurred. His parent presented, at age 65 years, slowly progressive memory deficits without behavioral impairment, and, on MRI, evidence of mesial temporal atrophy, consistent with a clinical diagnosis of Alzheimer disease (AD). CONCLUSIONS: This is the second family carrying the MAPT Q336H mutation reported so far. We showed that svPPA and AD-like phenotype can be associated with this mutation. A wide clinical variability exists at the intrafamilial level for Q336H MAPT mutation, pointing to genetic and/or environmental influencing factors on disease expression. We also confirmed that svPPA can be associated with MAPT mutations, suggesting that this gene should be analyzed also in patients with svPPA, especially with early onset. In addition, an AD-like phenotype may be associated with this mutation, suggesting its different effects on protein misfolding and aggregation.

Structural MRI Signatures in Genetic Presentations of the Frontotemporal Dementia/Motor Neuron Disease Spectrum.

BACKGROUND AND OBJECTIVES: To assess cortical, subcortical, and cerebellar gray matter (GM) atrophy using MRI in patients with disorders of the frontotemporal lobar degeneration (FTLD) spectrum with known genetic mutations. METHODS: Sixty-six patients carrying FTLD-related mutations were enrolled, including 44 with pure motor neuron disease (MND) and 22 with frontotemporal dementia (FTD). Sixty-one patients with sporadic FTLD (sFTLD) matched for age, sex, and disease severity with genetic FTLD (gFTLD) were also included, as well as 52 healthy controls. A whole-brain voxel-based morphometry (VBM) analysis was performed. GM volumes of subcortical and cerebellar structures were obtained. RESULTS: Compared with controls, GM atrophy on VBM was greater and more diffuse in genetic FTD, followed by sporadic FTD and genetic MND cases, whereas patients with sporadic MND (sMND) showed focal motor cortical atrophy. Patients carrying C9orf72 and GRN mutations showed the most widespread cortical volume loss, in contrast with GM sparing in SOD1 and TARDBP. Globally, patients with gFTLD showed greater atrophy of parietal cortices and thalami compared with sFTLD. In volumetric analysis, patients with gFTLD showed volume loss compared with sFTLD in the caudate nuclei and thalami, in particular comparing C9-MND with sMND cases. In the cerebellum, patients with gFTLD showed greater atrophy of the right lobule VIIb than sFTLD. Thalamic volumes of patients with gFTLD with a C9orf72 mutation showed an inverse correlation with Frontal Behavioral Inventory scores. DISCUSSION: Measures of deep GM and cerebellar structural involvement may be useful markers of gFTLD, particularly C9orf72-related disorders, regardless of the clinical presentation within the FTLD spectrum.

Emotional imagination of negative situations: Functional neuroimaging in anorexia and bulimia.

AIM: The present study aims to extend the knowledge of the neural correlates of emotion processing in first episode subjects affected by anorexia nervosa (AN) or bulimia nervosa (BN). We applied an emotional distress paradigm targeting negative emotions thought to be relevant for interpersonal difficulties and therapeutic resistance mechanisms. METHODS: The current study applied to 44 female participants with newly diagnosed AN or BN and 20 matched controls a neuroimaging paradigm eliciting affective responses. The measurements also included an extensive assessment comprising clinical scales, neuropsychological tests, measures of emotion processing and empathy. RESULTS: AN and BN did not differ from controls in terms of emotional response, emotion matching, self-reported empathy and cognitive performance. However, eating disorder and psychopathological clinical scores, as well as alexithymia levels, were increased in AN and BN. On a neural level, no significant group differences emerged, even when focusing on a region of interest selected a priori: the amygdala. Some interesting findings put in relation the hippocampal activity with the level of Body Dissatisfaction of the participants, the relative importance of the key nodes for the common network in the decoding of different emotions (BN = right amygdala, AN = anterior cingulate area), and the qualitative profile of the deactivations. CONCLUSIONS: Our data do not support the hypothesis that participants with AN or BN display reduced emotional responsiveness. However, peculiar characteristics in emotion processing could be associated to the three different groups. Therefore, relational difficulties in eating disorders, as well as therapeutic resistance, could be not secondary to a simple difficulty in feeling and identifying basic negative emotions in AN and BN participants.

Machine Learning Profiling of Alzheimer's Disease Patients Based on Current Cerebrospinal Fluid Markers and Iron Content in Biofluids.

Alzheimer's disease (AD) is the most common form of dementia, characterized by a complex etiology that makes therapeutic strategies still not effective. A true understanding of key pathological mechanisms and new biomarkers are needed, to identify alternative disease-modifying therapies counteracting the disease progression. Iron is an essential element for brain metabolism and its imbalance is implicated in neurodegeneration, due to its potential neurotoxic effect. However, the role of iron in different stages of dementia is not clearly established. This study aimed to investigate the potential impact of iron both in cerebrospinal fluid (CSF) and in serum to improve early diagnosis and the related therapeutic possibility. In addition to standard clinical method to detect iron in serum, a precise quantification of total iron in CSF was performed using graphite-furnace atomic absorption spectrometry in patients affected by AD, mild cognitive impairment, frontotemporal dementia, and non-demented neurological controls. The application of machine learning techniques, such as clustering analysis and multiclassification algorithms, showed a new potential stratification of patients exploiting iron-related data. The results support the involvement of iron dysregulation and its potential interaction with biomarkers (Tau protein and Amyloid-beta) in the pathophysiology and progression of dementia.

Genetic variation in APOE, GRN, and TP53 are phenotype modifiers in frontotemporal dementia.

Frontotemporal dementia (FTD) is a clinical, genetic, and pathologic heterogeneous group of neurodegenerative diseases. In this study, we investigated the role of APOƐ4, rs5848 in GRN, and rs1042522 in TP53 gene as disease risk factors and/or phenotype modifiers in 440 FTD patients, including 175 C9orf72 expansion carriers. We found that the C9orf72 expansion carriers showing an earlier age at onset (p < 0.001). Among the clinical groups, the FTD-MND (motoneuron disease) showed the lowest survival (hazard ratio [HR] = 4.12), and the progressive nonfluent aphasia group showed the highest onset age (p = 0.03). In our cohort, the rs1042522 in TP53 was associated with disease onset (p = 0.02) and survival (HR = 1.73) and rs5848 GRN with a significantly shorter survival in CC homozygous patients (HR = 1.98). The frequency of APOƐ4 carriers was significantly increased in the C9orf72 noncarriers (p = 0.022). Although validation of our findings is necessary, our results suggest that TP53, GRN, and APOE genes may act as phenotype modifiers in FTD and should be considered in future clinical trials.

Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis.

We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40-64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington's disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.

Neuropathological Alzheimer's Disease Lesions in Nasu-Hakola Disease with TREM2 Mutation: Atypical Distribution of Neurofibrillary Changes.

Nasu-Hakola disease is a rare autosomal recessive disorder associated to mutations in TREM2 and DAP12 genes, neuropathologically characterized by leukoencephalopathy with axonal spheroids. We report the neuropathologic findings of a 51-year-old female with a homozygous mutation (Q33X) of TREM2 gene. Beside severe cerebral atrophy and hallmarks of Nasu-Hakola disease, significant Alzheimer's disease lesions were present. Neurofibrillary changes showed an atypical topographic distribution being severe at spots in the neocortex while sparing the mesial temporal structures. Our finding suggests that TREM2 genetic defects may favor Alzheimer's disease pathology with neurofibrillary changes not following the hierarchical staging of cortical involvement identified by Braak.