Brain sagging dementia (BSD) is a rare but devastating form of early-onset dementia characterized by intracranial hypotension and behavioral changes resembling behavioral variant frontotemporal dementia. This review aims to provide a comprehensive overview of BSD, highlighting its pathomechanism, diagnostic tools, and available treatment options.BSD exhibits a complex clinical manifestation with insidious onset and gradual progression of behavioral disinhibition, apathy, inertia, and speech alterations. Additionally, patients may exhibit brainstem and cerebellar signs such as hypersomnolence and gait disturbance. Although headaches are common, they may not always demonstrate typical orthostatic features. Recent radiological advances have improved the detection of CSF leaks, enabling targeted treatment and favorable outcomes.Understanding the pathomechanism and available diagnostic tools for BSD is crucial for a systematic approach to timely diagnosis and treatment of this reversible form of early-onset dementia, as patients often endure a complex and lengthy clinical course. Brain sagging dementia (BSD) is a rare however devastating disorder that has recently gained increasing attention and recognition –7, 8•, 9In our 2022 systematic review, we aimed to provide a diagnostic tool to facilitate the detection and diagnosis of BSD ••. AlthoThis article offers a comprehensive insight into different aspects of BSD, drawing from current updated literature, our earlier publications on the topic, and our clinical experience in treating these patients at the CSF Disorder Division, Department of Neurosurgery, Oslo University Hospital. A better understanding of BSD, including its pathogenesis, clinical and radiological presentation, and available therapies, is crucial for timely diagnosis and effective management of this reversible form of early-onset dementia.spontaneous intracranial hypotension abbreviated SIH [The history of BSD can be traced back to post-spinal headache, which is associated with lumbar puncture , a technique described by German physician Heinrich Irenaeus Quincke in 1891 for diagnostic and therapeutic purposes . A few yated SIH , 17. TheIn 1998, Pleasure et al. presented the first case of SIH accompanied by brain sagging and behavioral and cognitive changes . In the The annual incidence of SIH is estimated at 5 cases per 100,000 individuals . The occAlthough CSF hypovolemia is a common factor in the pathogenesis of SIH and BSD, the latter involves additional mechanisms that make the disease more distinct and complex Fig. .Fig. 1ScOne of the crucial functions of CSF is to provide mechanical buoyancy and protect the floating brain by reducing its weight within the skull to less than 50 g . This buIn an adult, the craniospinal system contains approximately 150 ml of CSF in the ventricular and subarachnoid spaces, with one-third located in the cranial compartment and two-thirds in the spinal compartment , 24••. IWhen a CSF leak occurs, the craniospinal CSF volume decreases, leading to a pathological increase in compliance and a shift to the left on the pressure–volume curve . This alThe exact mechanism underlying the development of behavioral and cognitive changes in patients with BSD is not fully understood, but several hypotheses have been proposed ••. Most One theory suggests that mechanical stretching, as explained previously, may also affect the frontotemporal cortical structures and/or their circuits , 6, 7, 9Another theory is based on diminished cortical perfusion as shown in single-photon emission computed tomography (SPECT) studies . The intAdditionally, venous stagnation caused by stenosis due to a decreased angle between the vein of Galen and straight sinus contributes to impaired venous drainage and cortical hypoperfusion in the frontotemporal, parietal, and cerebellar cortical areas , 33.A third theory involves associated hypometabolism in the frontotemporal cortical region, which was illustrated by positron emission tomography (PET) in a study by Schievink et al. involving approximately three-fourths of BSD patients , 6. The Although the subdural fluid collection resulting from volume compensatory mechanism has also been suggested as a possible explanation for behavioral and cognitive changes, several lines of evidence suggest this to be an unlikely mechanism , 34–36. In essence, any condition that leads to CSF hypovolemia has the potential to cause intracranial hypotension and BSD. While various causes have been described in patients with SIH, such as total body water loss (true hypovolemic state), CSF shunt overdrainage, and following cranial surgery, these causes are rarely reported in BSD patients ••, 30. RIn BSD, the CSF leak appears to develop over a longer period of time ••, 2•. CThese findings support the notion that many BSD patients initially experience symptoms of SIH that are overlooked or not treated properly in the first place, allowing the disease to reach a chronic state where behavioral and cognitive changes manifest ••. It istype 1 CSF leak) [type 2 CSF leak) [type 3 CSF leak) [type 4 CSF leak [Based on these observations, it is reasonable to assume that the characteristics and magnitude of the CSF leak in BSD patients may differ to some extent from those in SIH patients. Recent or remote spinal trauma has been reported as an etiology of dural tear (SF leak)  and CSF SF leak) •, 39. EvSF leak) , 40, 41.SF leak) , spondylSF leak)  in BSD pSF leak) •. HoweveCSF leak .internal SIH in which intracranial hypotension occurs without an evident CSF leak, primarily due to pathologically increased spinal compliance that results in a cranial-to-spinal CSF shift and subsequent brain sagging [dural reduction surgery in cases where an extremely thin dura is identified rather than a real tear, or in cases of relapses despite repeated treatments [Goldberg et al. recently described a novel phenomenon called  sagging ••. This eatments , 43. Theeatments , 43.Traditionally, the focus on cranial causes of CSF leaks has been overlooked ••. We reA detailed medical history assessment and thorough interviews with both the patients and their relatives are essential in diagnosing BSD ••, 2•. TUp to 90% of patients with BSD experience some form of headache during the course of the disease ••. HowevBSD patients may also experience neck and back stiffness and pain. These symptoms may be associated with the site of CSF leak or result from tension caused by the headaches and the traction of cranial and dural structures.The gradual and insidious development of behavioral and cognitive changes is an absolute requirement for the diagnosis of BSD ••. TheseTheir decision-making abilities may be severely hampered, leading to reckless behavior, criminal acts, and even sexual assault , 53, 55.While BSD can lead to anterograde amnesia , 46, 48,Speech and language abilities may also be affected in BSD , 56, 59.Although both BSD and bvFTD share a gradual progression of symptoms, BSD tends to progress more rapidly (spanning at least over 3 to 4 weeks). This disparity is attributed to the distinct nature in pathomechanisms underlying these two conditions. The active CSF leak in BSD, causes more rapid structural changes and clinical deterioration compared to bvFTD .Additional distinguishing features between BSD and bvFTD lie in the clinical signs and symptoms associated with brainstem and cerebellar involvement in BSD patients. Up to 60% of BSD patients exhibit cerebellar symptoms, including dysarthria, ataxia, and an unsteady gait , 59–62.Bulbar manifestations and parkinsonism have also been reported in association with CSF leaks and BSD •, 56, 63Similarly, the direct compression of brainstem nuclei and strain on cranial nerves due to brain sagging can also cause diplopia, visual disturbance, dysarthria, dysphagia, and tinnitus , 59, 62.Although altered consciousness and coma have been reported in severe cases of CSF leak and SIH, it is important to distinguish these findings with symptoms of BSD.Coma resulting from severe CSF leak and brain sagging primarily arises due to the direct compression and distortion of brainstem reticular formation . In BSD,Hypothetically, both SIH and BSD with severe brain sagging can potentially lead to stupor and coma. However, the slow progression of symptoms in BSD enables patients to seek medical attention before reaching a critical stage of brain sagging that could result in coma. Moreover, reduced consciousness and coma can be caused by increased intracranial pressure when the critical threshold of the intracranial pressure volume curve is surpassed, which can occur due to subdural fluid collection, CSF outflow obstruction, and edema caused by venous stagnation •, 36. DiHypersomnolence is a common occurrence in many patients with BSD and is likely associated with the involvement of deep midline structures and the brainstem, including the reticular formation. While some authors suggest that hypersomnolence is an important hallmark of the syndrome and present in all cases , 4••, thContrast-enhanced MRI is considered the gold standard for diagnosing BSD , 66. TheAlthough pachymeningeal dural enhancement is considered a hallmark finding in SIH, only two-thirds of BSD patients exhibit this finding ••. A lacComputed tomography (CT) is typically the initial imaging modality used due to its availability. However, its diagnostic value in BSD is limited. Nonetheless, in acute settings, CT can be helpful in ruling out other overt pathologies . FurtherThe value of PET and SPECT in evaluating and diagnosing BSD is uncertain. Like CT, they may be useful in differentiating BSD from other diseases such as Parkinson’s disease and progressive supranuclear palsy (PSP); however, they do not reliably differentiate BSD from bvFTD. Hypometabolism has been observed in the frontotemporal region of BSD patients, similar to patients with bvFTD , 6. AlthIdentifying CSF leaks is generally challenging, particularly in patients with BSD. In fact, a CSF leak was only localized in a small proportion 13%) of BSD patients [3% of BSDSpine MRI has become the preferred imaging modality for identifying signs of CSF leak and should be the initial choice whenever possible . A fat-sIt is essential to bear in mind that CSF extravasation is sometimes demonstrated at cervical level 1–2 and in the cervicothoracic junction, which can be misleading as false localizing signs and do not accurately represent the CSF leak site , 76.type 2a CSF leak) [In cases where an obvious leak site or CSF accumulation in the spinal epidural space is absent, indirect pathologies indicative of the leakage source should be investigated. These may include conditions such as spondylotic spur, disc herniation, spinal meningeal diverticula, and signs of vascular abnormalities. In a series by Schievink et al., a spinal meningeal diverticulum (SF leak)  was founSF leak) . The preSF leak) ••. HowevSF leak) .type 3 CSF leak) [If the aforementioned measures fail to identify a CSF leak, more invasive methods such as myelogram with intrathecal contrast (either MRI or CT) or DSM should be considered , 73•. ThSF leak) , offerinSF leak) , 80•. ReSF leak) ••, 80•. SF leak) ••.2O) during spinal lumbar puncture was considered a primary diagnostic approach for SIH [Traditionally, measuring low opening pressure  is a form of early-onset dementia that can be reversible if diagnosed and treated effectively. While BSD shares similarities with SIH caused by a CSF leak, its pathogenesis involves additional anatomical and pathophysiological changes, resulting in a complex syndrome characterized by insidious and progressive behavioral and cognitive changes, headaches, and brainstem and cerebellar symptoms. This condition significantly impacts the daily activities of affected patients and places a considerable burden on both patients and their caregivers.Fortunately, with the increasing awareness and advancements in diagnostic workup of BSD, a growing number of patients are being diagnosed and properly treated with favorable outcomes. Given the rarity and complexity of BSD, a comprehensive understanding of its underlying mechanisms, diagnostic approaches, and treatment options is crucial in identifying and effectively managing these patients.


GDAP1 gene have been reported. However, despite implications for mitochondrial fission and fusion, cytoskeletal interactions, and response to reactive oxygen species, the etiology of GDAP1-linked CMT is poorly understood at the protein level. Based on earlier structural data, CMT-linked mutations could affect intramolecular interaction networks within the GDAP1 protein. We carried out structural and biophysical analyses on several CMT-linked GDAP1 protein variants and describe new crystal structures of the autosomal recessive R120Q and the autosomal dominant A247V and R282H GDAP1 variants. These mutations reside in the structurally central helices ⍺3, ⍺7, and ⍺8. In addition, solution properties of the CMT mutants R161H, H256R, R310Q, and R310W were analysed. All disease variant proteins retain close to normal structure and solution behaviour. All mutations, apart from those affecting Arg310 outside the folded GDAP1 core domain, decreased thermal stability. In addition, a bioinformatics analysis was carried out to shed light on the conservation and evolution of GDAP1, which is an outlier member of the GST superfamily. GDAP1-like proteins branched early from the larger group of GSTs. Phylogenetic calculations could not resolve the exact early chronology, but the evolution of GDAP1 is roughly as old as the splits of archaea from other kingdoms. Many known CMT mutation sites involve conserved residues or interact with them. A central role for the ⍺6-⍺7 loop, within a conserved interaction network, is identified for GDAP1 protein stability. To conclude, we have expanded the structural analysis on GDAP1, strengthening the hypothesis that alterations in conserved intramolecular interactions may alter GDAP1 stability and function, eventually leading to mitochondrial dysfunction, impaired protein-protein interactions, and neuronal degeneration.Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its subtypes are linked to mutations in dozens of different genes, including the gene coding for ganglioside-induced differentiation-associated protein 1 (GDAP1). The main GDAP1-linked CMT subtypes are the demyelinating CMT4A and the axonal CMT2K. Over a hundred different missense CMT mutations in the  Mitochotabolism . Since nvia fission and fusion, which are driven by proteins on the mitochondrial outer membrane (MOM), such as mitofusin 1 and 2 (MFN1/2), and the mitochondrial inner membrane (MIM), such as OPA1 and FIS1. Auxiliary proteins may bind to either MOM or MIM to enhance the process. The ganglioside-induced differentiation-associated protein 1 (GDAP1) is an integral MOM protein, proposed to have an auxiliary role in mitochondrial fission and fusion [via redox-dependent interactions with cytoskeletal components [Mitochondria are not isolated organelles, but interact with other cellular compartments, such as the endoplasmic reticulum, lysosomes, and peroxisomes, exchanging metabolites , 6, 7. Md fusion , possiblmponents . HoweverS-transferases (GST), and it contains unique flexible loops [in vivo. Members of the GST superfamily share a common fold, but structural differences and low sequence conservation result in a diverse group of substrates; hence, it is possible that GDAP1 is an enzyme, but the substrate and reaction mechanism remain unidentified.Structurally, GDAP1 resembles glutathione le loops , 11. GDAle loops , and while loops , the GDAGDAP1 gene is one of the most common missense mutation targets linked to CMT [Increasing numbers of genes related to mitochondrial function have been linked to neuropathophysiological conditions. Inherited polyneuropathies are a genetically and clinically diverse group of neurodegenerative diseases, which affect the outer motor and sensory neurons in the peripheral nervous system (PNS) , 15, thed to CMT , 21, 22.d to CMT , 23. Thed to CMT . In addid to CMT . In fullGSTs often contribute to mechanisms of neurodegenerative disease , 25. GST6 tag and a Tobacco Etch Virus (TEV) protease digestion site, for producing soluble recombinant human GDAP1 in E. coli, have been described [The GDAP1Δ303–358 and GDAP1Δ319–358 constructs, with an N-terminal Hisescribed . The poiE. coli BL21(DE3) using autoinduction [2+-NTA chromatography, and the affinity tag was cleaved using TEV protease. Another Ni2+-NTA affinity step removed the tag and TEV protease. Size exclusion chromatography (SEC) was performed on a Superdex 75 10/300 GL increase column (Cytiva) using 25 mM HEPES (pH 7.5) and 300 mM NaCl (SEC buffer) as mobile phase. SEC peak fractions were analysed with SDS-PAGE and concentrated by centrifugal ultrafiltration.Recombinant GDAP1 variants were expressed in nduction , and purnduction . BrieflyDL-malic acid (pH 7.3), 20% (w/v) PEG3500. R282H crystals were obtained in 0.1 M succinic acid, 20% (w/v) PEG 3350. For cryoprotection, crystals were briefly soaked in a mixture containing 10% PEG200, 10% PEG400, and 30% glycerol, before flash cooling in liquid N2.Mutant GDAP1Δ303–358 crystals were obtained using sitting-drop vapour diffusion at +4°C. Proteins were mixed with mother liquor on crystallisation plates using a Mosquito LCP nano-dispenser (TTP Labtech). The protein concentration was 10–30 mg/ml in 75 nl, and 150 nl of reservoir solution were added. R120Q crystals were obtained in 0.1 M succinic acid, 15% (w/v) PEG 3350. A247V crystals were obtained in 0.15 M Table 1, and the refined coordinates and structure factors were deposited at the Protein Data Bank with entry codes 7B2G (R120Q), 8A4J (A247V), and 8A4K (R282H).X-ray diffraction data were collected at the PETRA III synchrotron  on the P11 beamline , 30 and A model for full-length human GDAP1 was obtained from AlphaFold2 . In addiq-range of 0.003–0.5 Å−1. SAXS data analysis, processing, and modelling were done in ATSAS 3.0 [ab initio models were generated using GASBOR [The structure and oligomeric state of the GDAP1 mutants were analysed with SEC-coupled small-angle X-ray scattering (SAXS). SEC-SAXS experiments were performed on the SWING beamline   .Synchrotron radiation circular dichroism (SRCD) spectra were collected from 0.5 mg/ml samples on the AU-SRCD beamline at the ASTRID2 synchrotron . The samples were prepared in 10 mM HEPES pH 7.5 and 100 mM NaF buffer, equilibrated to room temperature in 0.1 mm closed circular quartz cuvettes . SRCD spectra were recorded from 170 nm to 280 nm at +25°C. Three scans per measurement were repeated and averaged. The spectra were processed using CDToolX .NanoDSF experiments were done at 0.5 mg/ml, and 3 aliquots of each sample were run simultaneously . Tryptophan fluorescence was excited at 280 nm, and emission was recorded at 330 and 350 nm, while the samples were heated from +20 to +90°C at a rate of 1°C/min. Changes in the fluorescence ratio (F350/F330) were used to determine apparent melting points. The sample analysis was done using Nanotemper PR.ThermControl analysis package utilizing Boltzmann fit analysis and average of three F350/F330 inflection points was taken. The curves presented in the figures are the averaged curves. The data were analyzed using Origin .Thermal stability of GDAP1 variants was studied by nanoDSF using a Prometheus NT.48 instrument (NanoTemper), in SEC buffer. e-value of 10−99 or less. In a second iteration, sequences with e-value < 10-7 were accepted, resulting in 5986 sequences. This initial set was divided into three subsets according to their annotations: (a) GDAP1, (b) GDAP1L1 and (c) GST-labelled. For many calculations, GDAP1 and GDAP1L1 were combined to improve the statistics of conservation calculations, as they form a clear subgroup within GST-like sequences. Since the separation was based on annotations, sequences labelled as hypothetical or putative were removed. Sequence calculations were done either on subgroups or across the full set of 5065 sequences.The NCBI non-redundant protein database was used for all sequence searches. Starting from the human GDAP1 reference sequence (NP_061845.2), iterative PSI-BLAST  searchesAll multiple sequence alignments were calculated with MAFFT . Resultsi in a multiple sequence alignment was given byjp is the probability of seeing amino acid type j at the site and the summation runs over the 20 amino acid types. Gaps were treated as missing data. The use of base 20 in the logarithm ensures that S ranges from 0 (fully conserved) to 1 (random). In the interpretation below, S<0.2 was considered conserved and S<0.1 as highly conserved.Per-site entropy at site KLD) captures the difference between two distributions. With discrete distributions (types of amino acids), the value at position i is given byi,jp and i,jq are the frequencies (probabilities) of seeing residue type j at position i in the first and second protein groups [The most interesting sequence sites are those which are conserved within groups of proteins, but different between groups. The Kullbach-Leibler divergence  [Maximum likelihood phylogenetic trees were calculated with MrBayes , 54 withe (iTOL)  using a Sequence data sets for phylogeny were different to those used for conservation calculations, the aim being to cover sequences from GDAP1 to common GSTs and archaea. The first sequence searches and guide trees had identified the archaeal and bacterial sequences closest to GDAP1. The similarity matrix suggested that bacterial GSTs were remotely related to both the eukaryotic and archaeal sequences. The first 799 database hits of GDAP1 formed one group, and the first 1000 hits from human GDAP1L1 (NP_001243666.1) the next. The first 1000 hits from the bacterial GST (TNE50161.1) and the human GST (sequence from PDB entry 1PKZ) were the third group. Finally, the first 1000 hits for the archaeal sequence (MAE98075.1) formed a fourth group.Each of these four sets were then reduced to 100 representatives based on the similarity matrix calculated during a sequence alignment . The proFig 1A and 1B.Building upon earlier work on GDAP1 structure –12, we fThe majority of CMT-linked missense mutations in GDAP1 are located within the vicinity of the hydrophobic clusters of the GST-like domains and the dimer interface , and thevia the α6-α7 loop, and Cys240 in this loop–itself being a CMT mutation site–is central to many interactions. The α6 helix can either extend or turn back towards the dimer interface, as seen in earlier crystal structures and models [Fig 1C). Open/close movements of α6 can be functionally relevant for GDAP1 interactions with other proteins, such as cofilin and tubulin [Fig 1D) and non-polar contacts, and many CMT-linked missense mutations are found on these helices .The GST-like core fold of GDAP1 is supported by the α7 helix, surrounded by helices α3, α6, and α8. The helix α3 is connected to α6 d models . A new m tubulin . The α8 There are 15 designated hydrogen bond contacts or ionic interactions between helices α3, α6, α7, and α8. Helix α7, which is central to the GDAP1 fold, makes only a few hydrogen bonds to the surrounding helices, and hydrophobic residue clustering houses the α7 helix in the core of the fold. This is in line with earlier centrality analyses of the GDAP1 fold, showing helix α7 to be the most central part of the 3D structure . Some ofTable 1).We have previously analysed the effects of R120W and H123R at the protein level, both residing on helix α3 [Fig 2A). This is further linked to alterations in surrounding side chain conformations.The R120Q mutation is located close to the N-terminal end of the α3 helix. Due to the mutation, residue 120 loses contact with the α6-α7 loop tip at Cys240, whereby the hydrogen bond between the Arg120 side chain and the backbone carbonyl of Cys240 is lost .The A247V mutation site resides near the N terminus of helix α7, tightly surrounded by helix α3 and the α6-α7 loop, and having van der Waals contacts to Val121, Tyr124, Cys240, and Thr245. Comparing to the wild-type protein, changes in the crystal structure are small, but an overall movement of surrounding protein segments is caused by the presence of Val in this position, due to the increased volume of the side chain. Both helices α3 and α6 move slightly away, without altering hydrogen-bonding patterns . All these interactions are lost upon the mutation, and the His residue in the mutant is observed in a double conformation, making no side-chain hydrogen bond contacts.R282H is located on helix α8, and the side chain of Arg282 points inwards in the wild-type GDAP1 structure; it is stacked against Trp238 and makes hydrogen bonds to the backbone carbonyl groups of residues 236 and 237 in the α6-α7 loop . All variants in this construct background eluted mainly as dimers, and the top of the peak was selected for the analyses. The largest deviation was observed for R161H, which had a larger Rg than the other variants, suggesting a more open structure . Also R120Q had a slightly different conformation, most clearly seen in the distance distribution. The pair distance distribution function showed that the maximum particle dimension in all samples was ~90–110 Å, which corresponds to a dimer . Ab initio modelling, here carried out on the R282H mutant, which was measured at the highest concentration, supported the presence of a dimeric GDAP1, corresponding to the conformation of the dimer we previously showed to fit the solution SAXS data for wild-type GDAP1 [Fig 3C). As the SEC-SAXS experiments were carried out at the maximum concentration for each variant, we complemented these experiments with a set of batch-mode SAXS experiments with wild-type GDAP1 and four mutant variants . All variants in this supplementary experiment are predominantly dimeric in the concentration range 1–4 mg/ml.The SAXS analysis of GDAP1Δ303–358 showed that the protein particle dimensions in solution correspond to a dimer, and the scattering profiles showed only minor shape differences, ruling out large-scale conformational differences or aggregation (Table 2). This behaviour has previously been linked to protein concentration, in that GDAP1 monomers can be observed at low concentration [Fig 3C), reflecting the presence of the C-terminal amphipathic domain in the construct. Comparing the Kratky plot for all variants studied here, it is evident that they all are rigidly folded, with little flexibility .For the two mutations affecting Arg310, we used the longer construct GDAP1Δ319–358, and both R310Q and R310W eluted as two peaks in SEC-SAXS, corresponding to a dimer and monomer . The nanoDSF experiment (Table 3) revealed a ~1–12°C decrease in apparent melting temperature for all core domain mutants, compared to the wild-type GDAP1Δ303–358 . This is similar to the previously studied R120W and H123R mutants, which had melting temperatures ~52–54°C, i.e. a drop of ~10°C compared to the wild-type protein [350/F330 ratio varies between the variants at the starting point, especially so for H256R, which may be an indication of subtle differences in structure. All in all, the biophysical data indicate that the structural effects of all studied CMT variants in the core domain are local, and the mutations do not cause large-scale conformational changes at a level detectable with SRCD or SAXS. However, all studied mutations in the core domain caused a decrease in thermal stability, suggesting a breakdown of stabilising intramolecular interactions within the GDAP1 molecule. As the most dramatic example, the mutation A247V inside the folded core decreased the stability by 13°C, indicating the importance of a correctly packed hydrophobic core for GDAP1 stability.For thermal stability and secondary structure analysis, nanoDSF and SRCD were performed. The SRCD spectra showed similar secondary structure composition for all variants and wild-type GDAP1, indicated by the fact that the spectral shapes were nearly identical, despite some fluctuation in amplitude. The latter could be caused by errors in concentration determination or aggregation status in the strong synchrotron UV beam, or by differences in dynamics of the secondary structure elements. The SRCD spectrum for R161H showed a slight change in the spectral shape between 205–225 nm, which could be linked to its more open conformation observed in SAXS above. It can be concluded that none of the studied mutations interfere strongly with the overall folding of GDAP1 . This can be explained by the fact that the segment carrying these mutations is no longer part of the core domain, but rather likely to represent an ⍺ helix attached to the membrane surface, possibly linking the membrane to the core domain. Overall, GDAP1 stability correlates with the dimer-monomer ratio observed in solution; the most destabilised mutants also show a higher fraction of monomeric protein on non-reducing SDS-PAGE , even though in the 3D structure, they are far from the dimer interface. The exact mechanism for this observation is currently unknown, but it could mean that altered protein dynamics and local conformations affect the dimer formation and/or that dimerisation in turn stabilises the protein fold.For the mutations at position 310, it was observed that both R310Q and R310W in fact increased the stability of GDAP1Δ319–358 by ~1°C , and the mapping shows that the residues pointing towards the dimer interface have only minor structural variations compared to the wild-type protein. In contrast, at the C-terminal end of helix α6, in all mutant crystal structures, the variation of the Cα atoms is high . This suggests that flexibility of helix α6 could arise from altered intramolecular contacts in the vicinity. The C-terminal end of the α6 helix, the conformation of which apparently is affected by the mutations studied here, is itself a target for multiple CMT mutations, affecting Gln218, Va219, Glu222, Arg226, and Glu227 [The studied mutations cause subtle variations in hydrogen bond and van der Waals distances in nearby residues in the crystal structures, while there are no drastic structural differences, when the mutant structures are superposed onto the wild-type crystal structure. However, when comparing the variation of the residues in helices α3, α6, α7, and α8, in comparison to the wild-type Cα-atoms, a specific pattern arises. We analysed the residues participating a hydrogen-binding network in the mutant structures . In essence, all substitutions to Arg120 are unfavourable; this shows that the interactions made by Arg120 are important for folding and stability. This is in line with our experimental data for both R120Q and R120W, which indicate minor changes in structure, but destabilisation of the fold. For Ala247, a mutation into Val is predicted to be slightly stabilising, indicating that such a replacement, in a tightly confined pocket and with potential long-range effects, is difficult for the prediction algorithm. In the case of Arg282, mutation to His is indeed one of the most destabilising variants in the prediction, linked to a loss of several key interactions by the Arg282 side chain.While the above analyses indicate an average effect that may destabilise GDAP1 structure, we looked at predictions for each mutation crystallised here in more detail . Intriguingly, based on large-scale sequence alignments, GDAP1 turns out to be more closely related to prokaryotic GSTs than eukaryotic proteins. In this situation, we must remember that sequence conservation across the GST superfamily is overall low. The phylogenetic calculations do not resolve to a clear bifurcating consensus tree, and the uncertainty is very high around the divergence of the GDAP1 group. One can say that GDAP1 is at least as far from generic eukaryotic GSTs as it is from prokaryotic proteins. The analysis provides little clues as to any enzymatic function of GDAP1, but it supports the findings that it has no conventional GST activity.The first calculations focussed on GDAP1  despite sampling over millions of MCMC steps. Fig 6D shows the last level of the phylogeny, where the consensus does converge to a proper bifurcating tree. Towards the right side, there is a branch of bacterial GST sequences (purple), and in the left branch, the GDAP1/GDAP1L1 sequences are found again. The figure also shows that orthologs of GDAP1 and GDAP1L1 are found in mammals, birds, amphibians, and fishes and likely orthologs of those genes in invertebrates as well.One may well want to interpret the results in terms of the endosymbiotic theory and remember that archaea are closer to eukaryotes than bacteria . Since GFig 7A). 44 residues were found with an entropy score ≤ 0.10 (highly conserved) and a fraction of non-gap characters in the alignment > 70% (Table 4); the entropy is mapped onto the GDAP1 structure in Fig 7B. The data highlight several interesting residues, which are known to be sites of CMT mutations, relevant for folding and structure, or with possible functions in ligand binding. These aspects are discussed more below.Sequence conservation (entropy) calculations were carried out on the different groups to identify their conserved residues and sites which differ between the groups. We first consider the GDAP1 subset of sequences . This suggests that they could be important for ligand binding and that mutations in these positions could affect GDAP1 function; indeed, Trp238 and Arg282 are CMT mutation sites. These 3 residues cluster on the side of GDAP1, which must face the MOM surface. One option is that the bound fatty acid, in fact, mimics the membrane surface, and that these residues are directly involved in membrane surface binding at the outer surface of the MOM.One intuitive approach to search for functionally important sites is to focus on residues involved in ligand binding, th GDAP1 . Gln235,Table 5 shows the entropy calculated for a combined data set . Since the GST superfamily is so diverse, we identified sites with entropy < 0.2 . The 14 sites that are conserved and present in at least 70% of the sequences are listed in Table 5. These include Leu239 and Gly241 in the ⍺6-⍺7 loop and Ala247 and Asp 248 on helix ⍺7. The effect of the A247V mutation on protein stability may have been therefore expected based on its conservation across this very broad set of GSTs.Table 6 shows the sites with a high KL divergence. These are those which are conserved in at least one group and different in the second group. The results have not been filtered by ignoring gaps since an insertion or deletion is of interest. Of the GDAP1 residues Cys51, Gly83, Tyr124, and Glu228 that are not assigned to a gap symbol, Cys51 and Gly83 are located close to the dimerisation site of GDAP1. Gly83 is a target of a non-pathological polymorphism, G83A [sm, G83A . The β sOn the other hand, Tyr124 and Glu228 in GDAP1 are close to each other in 3D space, being located on the core helices ⍺3 and ⍺6, respectively. The conversion of tyrosine to tryptophan (Tyr124 → Trp) and glutamic acid to glutamine (Glu228 → Gln) between GDAP1 and canonical GSTs causes only small changes in physicochemical properties. However, the central location of these residues in the GDAP1 fold suggests this finding may reflect a structural or functional aspect specific to GDAP1.Of additional interest are Lys193 and Asn166, which are both close to the GDAP1-specific insertion. In contrast to the crystal structure, where Asn166 is not visible, the AlphaFold2 model predicts that the helix ⍺6 is bent, which brings Lys193 and Asn166 into close proximity. This could enable intramolecular interactions or provide a specific site for protein-protein interactions. Overall, the results indicate that certain subdomains in GDAP1 may have evolved to fulfill a different function compared to canonical GSTs.Mutations in dozens of genes are causative for various subtypes of CMT disease. For most of them, the effects at the molecular level are not known, but one could even say that the working of the native protein is often poorly understood. One of the characterised examples is myelin protein P2, which loses thermal stability upon all the known 6 CMT mutations in the protein, while the crystal structure remains nearly unaltered , 58. FurMutations in the GST-like domain of GDAP1 have a broad pathological spectrum. The molecular basis remains unknown despite cellular observations confirming the causality of impaired mitochondrial dynamics. Accurate structural information is required to support these findings. Below, we shall discuss some implications of our findings to understand GDAP1 function at the molecular level and the effects of missense mutations therein.GDAP1 gene compared to other CMT target genes, especially when the size of the protein is taken into consideration [CMT-linked missense mutations are relatively common in the deration , 71. WitOf the residues highlighted by the entropy analysis either within the GDAP1 sequence set or the entire GST set, several are targets for missense mutations. Here, we shall briefly compare selected CMT mutations in GDAP1. The immediate environment of each residue is considered to shed light on local effects of each mutation.Tyr29 is conserved at the symmetry axis of the GDAP1 dimer, forming a H bond between OH groups of Tyr29 from the two protomers. Y29S  would boLeu239 is at the tip of the ⍺6-⍺7 loop, inserted into the structural core, and its mutation to Phe has been reported in CMT . The sidAla247, studied in the current work, lies centrally in the GDAP1 fold on helix ⍺7. A247V is linked to CMT, showing that no larger residue fits into this tight space and A247V destabilised the GDAP1 fold. Ala247 is one of the most conserved residues in the GST superfamily, indicating a role in the GST fold. Other residues conserved in the GDAP1 set include Leu255 and Gly262, which lie in the middle and at the end of helix ⍺7, respectively. L255F  would beArg282, studied here in the form of the CMT mutation R282H, is one of the most conserved residues in the GDAP1/GDAP1L1 subfamily. Its strong hydrogen bonding to the carbonyl groups in the ⍺6-⍺7 loop indicates a central role in GDAP1 structure. In addition to R282H, the mutation R282C has been reported , and we Fig 8). Interestingly, Arg310 resides in the segment originally coined the hydrophobic domain; in our view, this fragment corresponds to an amphipathic helix, which could bind onto a phospholipid membrane surface. The AlphaFold2 prediction supports this view. Arg310 is likely to link this membrane-bound helix to the folded core of GDAP1 and, therefore, directly affect the conformation of full-length, membrane-bound GDAP1. This is supported by the lack of a destabilising effect on recombinant GDAP1 structure in our experiments; Arg310 is located outside the folded core and has a more subtle role.Arg310 is not present in the constructs we used for crystallisation, and hence, we have no high-resolution data on its conformation. However, the AlphaFold2 model of GDAP1 extends our crystal structures and predicts that Arg310 points away from the MOM surface and forms two salt bridges to acidic residues in the GDAP1 fold . In GDAP1, the 3 residues at the tip of the loop (238–240) are all targets for CMT disease mutations, highlighting the crucial importance of this segment. Our entropy analysis confirms the strong conservation of this segment. We can then take a broader scope and further look at residues interacting with the ⍺6-⍺7 loop in GDAP1. Of note, several of the residues in direct contact with the ⍺6-⍺7 loop are known CMT mutation target sites and/or highly conserved (Table 4). Hence, the inward-bending ⍺6-⍺7 loop appears to be a central structural feature in GDAP1, and its alteration either directly or via disturbance of intramolecular interactions may be a common mechanism for CMT.The ⍺6-⍺7 loop is a central feature in the structure of GDAP1, but also in all canonical GSTs. This loop inserts itself back into the protein core, being a central interaction hub between helices ⍺3, ⍺6, ⍺7, and ⍺8  in the GST superfamily. While the entire evolutionary pathway cannot be traced based on the analyses, it is intriguing that GDAP1 is closer to bacterial than eukaryotic GSTs. It is possible that during evolution, GDAP1 has lost the characteristic GST activity, while becoming an integral membrane protein of the MOM. Its functions could, therefore, be mediated through protein-protein interactions instead of enzymatic activity. The functions can be redox-regulated, which could explain the observation that the human GDAP1 dimer is mediated by a disulphide bridge ia Cys88 .S. japonicum [Fig 10A).To complement the above analyses, we superposed crystal structures of human GDAP1 and a canonical GST, that from aponicum , and anaFig 10B). These observations highlight the importance of the central ⍺6-⍺7 loop in intramolecular interaction networks and fold stability of GDAP1.Arg120 in GDAP1 is a target of several CMT mutations –84, and per se, appears surprisingly conserved in the GST superfamily in our dataset (Table 4). In the superposed individual GST structure , this residue is Pro, which fits well into the structure due to slightly different conformations of the main helices in GST. In GDAP1, Ala247 is so snugly packed  that even the addition of two methyl groups in the A247V variant causes protein instability and disease, despite only minor effects in the crystal state. A247V is an example of a mutation introduced into the hydrophobic core that may have long-range effects on the entire protein structure stability. One of the residues in GDAP1 highlighted by the KL divergence analysis, Tyr124, closely interacts with Ala247, indicating a GDAP1-specific arrangement at this site. Additionally, one of the most conserved GDAP1 residues, Asp248, is central in a hydrogen bonding network linking the conserved CMT target Tyr279 into the picture .Ala247 of GDAP1, although perhaps a mundane residue Fig 10D). This is another example of an Arg–loop backbone interaction in GDAP1 that is both conserved and relevant for human disease mutations.GST has an Arg corresponding to Arg282, and this residue is highly conserved. Arg282 in GDAP1 interacts directly with the backbone of the ⍺6-⍺7 loop, and a similar interaction is observed in GST Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


Background and Objectives: The prevalence of type 2 diabetes mellitus in adolescents has increased rapidly in recent decades. However, the role of adipokines on pathophysiology in young-onset type 2 diabetes mellitus (YDM) is not clear. In this article, we explored the relationships between the adipokines (visfatin and retinol binding protein 4 (RBP4)) and metabolic syndrome (MetS) components in both YDM and late-onset type 2 diabetes mellitus (ODM). Materials and Methods: There were 36 patients with YDM (23.6 ± 4.8 years) and 36 patients with ODM (54.3 ± 10.1 years) enrolled. Visfatin, RBP4, and MetS components were measured. The relationships between visfatin, RBP4 and MetS components were assessed in YDM and ODM. Results: The visfatin, but not the RPB4 level, was significantly higher in YDM than in ODM. After adjusting for age and body mass index, visfatin was not related to any MetS components except that there was a negative correlation with fasting plasma glucose (FPG). As for RPB4, triglyceride was found to be positively and FPG negatively related to RBP4 in YDM. However, in ODM, the only positive relationship that existed was between RBP4 and diastolic blood pressure. Conclusions: In conclusion, both visfatin and RBP4 had certain roles in diabetes and MetS although their relationships were different in YDM and ODM. Further studies are needed to explore their physiological and pathological effects in glucose metabolism. The prevalence of young-onset type 2 diabetes mellitus  has been rising dramatically in recent decades worldwide. This is probably due to the parallel increase in the prevalence of obesity in children . Thus, tAn important feature of obesity is an accumulation of excessive adipose tissues in subcutaneous and visceral regions. In recent years, adipose tissue is regarded as an endocrine organ that secretes a variety of cytokines regulating metabolism, reproduction, inflammation, and cardiovascular function . Pre-B cAtherosclerosis Risk in Communities Study (ARIC Study) found this association between RBP4 and the onset of T2D only in women, not in men [At the same time, retinol binding protein 4 (RBP4) is highly expressed in adipose tissue and found to be higher in subjects with obesity and T2D . This mit in men . SimilarThe understanding of the different pathogenesis between YDM and ODM is crucial for reference of preventive strategies on YDM in the future. So far, there is no study investigating the roles of visfatin and RBP4 between YDM and ODM. Comparing their relationships with MetS components between YDM and ODM becomes important. In the present study, we tried to explore and compare the circulating visfatin and RBP4 levels between YDM and ODM, and their relationships with MetS components.We enrolled patients with T2D who visited the outpatient clinic of a local hospital in Taipei, Taiwan. The diagnostic criteria of T2D were based on the ADA guideline (fasting plasma glucose ≥ 7 mmol/dL). All subjects were being treated with only oral hypoglycemic agents at the time of enrollment. Subjects who received insulin injection or experience diabetic ketoacidosis or the presence of major cardiovascular, respiratory, renal, or endocrine disorders were excluded. Subjects who took any medication, except for oral hypoglycemic agents, known to affect the metabolism of glucose and lipid were also excluded. The dose of oral hypoglycemic agents was maintained at least three months prior to the study. In addition, subjects who refused to participate or had missing or incomplete data were excluded. The definition of the YDM group was under 22 years old at the time they were enrolled in the study. On the other hand, for the ODM group, it was more than 40 years old. For patients in the ODM group, their annual health exam or medical records were reviewed for assurance of T2D onset age of more than 40 years. The flowchart of participant selection is displayed in 2 (kg/m2). Waist circumference (WC) was taken at the midway point between the inferior margin of the last rib and crest of the ilium in a horizontal plane and measured to the nearest 0.1 cm. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by nursing staff using standard mercury sphygmomanometers on the right arm of seated participants who had rested for 5 min. Blood samples were drawn from an antecubital vein for biochemistry analysis.Subjects received a stable diet three days before they entered the study. After overnight fasting, subjects visited our clinic at 8 am. Information on medical history was obtained through interviews by the senior nursing staff. A complete physical examination was done and the body mass index (BMI) was calculated as body weight/body heightPlasma was separated from the blood within one hour and stored at −30 °C until analyzed. Circulating visfatin was measured by commercial enzyme immunoassay kits . The intra-assay and inter-assay coefficients of variation were <10% and <15%, respectively, for visfatin. RBP4 levels were determined by the Human Retinol-Binding Protein 4 ELISA Kit . The intra-assay and inter-assay coefficients of variation were 3.8% and 9.5%, respectively, for RBP4. Fasting plasm glucose (FPG) was measured by a glucose oxidase method and analyzer . Hemoglobulin A1c (HbA1c) was measured by a Bio-Rad Variant II automatic analyzer . Triglyceride (TG) was measured using the dry, multilayer analytical slide method in the Fuji Dri-Chem 3000 analyzer . HDL-C concentration was analyzed using an enzymatic, cholesterol assay method following dextran sulfate precipitation.t-test was used to evaluate visfatin, RBP4, HgbA1c, and MetS components between subjects with YDM or ODM. Because RBP4 and visfatin are tightly related to adiposity, the relationships of RBP4, visfatin, and MetS components were assessed by Pearson’s correlation analysis after adjusting for age and BMI. For further analysis, visfatin and RBP4 were analyzed by multivariate linear regression with age, BMI, and all of the MetS components. All statistical tests were two-sided, and p values < 0.05 were considered to indicate statistical significance.Statistical analysis was performed by using SPSS . Continuous variables were expressed as the mean ± standard deviation. An independent Student Finally, 36 patients with YDM (23.6 ± 4.8 years) and 36 patients with ODM (54.3 ± 10.0 years) were enrolled. The demographic characteristics and MetS components in YDM and ODM are shown in p = 0.085) in YDM and DBP  in ODM. However, visfatin was still positively related to TG  and negatively related to FPG  in ODM after multivariate linear regression.After adjustment of age and BMI, the correlation of visfatin and RBP4 with MetS components in YDM and ODM are shown in To the best of our knowledge, this is the first paper exploring the visfatin and RBP4 levels between YDM and ODM. We found that the visfatin levels are significantly lower in ODM. However, the same difference is not noted in RBP4. After adjustment of age and BMI, visfatin is not related to any components of MetS in YDM, but it was negatively related to FPG and positively to TG in ODM. As for RBP4, our results showed that it is positively related to TG in YDM and to DBP in ODM.In the present study, we have found that HbA1c, TG were higher and HDL-C were lower in YDM. These results are in line with other mainstream reports. For instance, Ahmad et al. reported similar findings that younger diabetes (<60 years old) patients had higher plasma glucose and lipid levels . AlthougIt is well-known that, compared with the subcutaneous adipose tissue, visceral adipose tissue contributes more to the risk of atherosclerosis and secretes pro-inflammatory cytokines including visfatin. Interestingly, visfatin is also found to be secreted by a variety of cells , includiNot surprisingly, visfatin secreted from visceral adipocytes is positively related to TG. Many studies also showed the same relationship between visfatin and TG ,36, whicThe roles of RBP4 in T2D have been of interest. Gavi et al. explored the 48 young and 55 elderly health subjects and compared RBP4 levels with MetS components and insulin sensitivity in different age groups . They foSeveral clinical investigations showed that higher RBP4 levels in subjects are related to hypertension ,43 and aThere are some limitations in our study. First, the number of subjects in our study is relatively small because the prevalence of patients with YDM (onset age < 22 years) is lower than in ODM. According to the Taiwan Nationwide Health Insurance database, the prevalence of YDM age of 18–22) was less than 0.5% in Taiwan , which i–22 was lIn conclusion, a higher visfatin level was noted in patients with YDM, and it was positively related to TG and negatively to FPG only in ODM. At the same time, RBP4 was positively associated with TG in YDM and DBP in ODM. The roles of both adipokines are important and need further study to clarify.


Amyotrophic lateral sclerosis (ALS) carries a uniformly poor prognosis with a median survival of 2–5 years after onset.The ALS field has been constrained by a lack of effective biomarkers, including diagnostic, prognostic, and pharmacodynamic tools, but that landscape is rapidly evolving.In the current study, Vu and colleagues report the results of unbiased proteomics analysis on longitudinal cerebrospinal fluid (CSF) samples from fast versus slow progressing patients and a mathematical model for the prediction of ALS progression rate.Limitations of the study include the modest number of patients, restricted by the resource‐intensive nature of discovery proteomics as well as the availability of longitudinal CSF samples. Because NfL was not among the most stringent list of 59 markers in the discovery cohort, the potential contribution of NfL to the sensitivity and specificity of the panel remains to be determined. Interestingly, RBP4, SERPINA4, and F12 are not CNS‐specific proteins, and thus the central versus peripheral origin of the increased CSF levels remains uncertain. Future analysis in paired plasma/CSF sample sets is needed to further clarify the potential pathophysiologic significance of these markers.Perhaps the most intriguing aspect of the study is the mathematical model, which raises the possibility that overall dysregulation of CSF protein networks may better predict ALS progression than changes in individual CSF markers. The feasibility of translation of such an approach to the clinic is uncertain, and “CSF entropy” remains to be compared to NfL as the reigning gold standard. However, it is tempting to consider chaos itself as a fitting benchmark for the formidable clinical heterogeneity of ALS.The authors declare no competing interests.


Embora o Teste da Fala (TF) seja um teste confiável e de baixo custo, seu uso para prescrição de exercício aeróbio ainda é limitado. Analisar a frequência cardíaca (FC) dos estágios do TF e no pico do teste de caminhada de 6 minutos (TC6min) como parâmetro para a prescrição de exercício aeróbio comparando com a FC no primeiro e segundo limiares ventilatórios (LV1 e LV2) do teste cardiopulmonar de exercício (TCPE). Pacientes com doença cardiovascular compareceram a 3 dias de avaliação: 1) anamnese e TCPE; 2) TC6min; e 3) TF. Foram usados ANOVA unidirecional de medidas repetidas ou teste de Friedman para comparar a FC no LV1 e LV2 com a FC nos estágios do TF: último positivo (TF+), primeiro equívoco (TF±) e negativo (TF−) e no pico do TC6min. O teste de Pearson ou Spearman avaliou se há correlação entre FC em LVs, estágios do TF e TC6min. A significância estatística foi fixada em 5%. O estudo incluiu 22 pacientes cardíacos . A FC no LV1 foi semelhante à FC no TF+  e TF± , e moderadamente correlacionada com o TF+ . A FC no LV2 foi semelhante ao TF− , com forte correlação . A FC no pico do TC6min foi significativamente diferente da FC no TF+, TF± e LV1 , mas semelhante ao TF− . A FC no TF+ e TF− reflete a FC no LV1 e LV2, respectivamente, diferentemente do TC6min, que foi semelhante apenas ao LV2. O TF pode ser um teste objetivo para auxiliar a prescrição de exercício aeróbio na reabilitação cardíaca. A prática adequada de exercício físico visa reduzir a pressão arterial, o risco de eventos cardiovasculares e o risco de mortalidade em pacientes cardiovasculares.2O principal objetivo da reabilitação cardiovascular é melhorar a aptidão cardiorrespiratória por meio do exercício aeróbio (EA).1 A análise do TCPE permite a identificação das variáveis mais adequadas e recomendadas, como consumo máximo de oxigênio, carga de trabalho, frequência cardíaca (FC) máxima, FC de reserva e limiares ventilatórios (LVs).5 No entanto, esse teste ainda tem acesso limitado, principalmente em países em desenvolvimento, devido aos altos custos, à necessidade de profissionais altamente capacitados e às análises complexas.5O teste cardiopulmonar de exercício (TCPE) é o teste de referência para avaliar a função cardiorrespiratória e prescrever a intensidade do EA.6 Outros métodos utilizam valores de FC absolutos ou relativos obtidos durante testes submáximos, como o teste de caminhada de 6 minutos (TC6min)9 e o teste da fala (TF).11 O TF é um teste validado e acessível, baseado em um protocolo de carga incremental e utiliza a percepção do conforto de fala como marcador da intensidade do exercício.13 Recentemente, o TF tem sido recomendado para avaliar a capacidade funcional e prescrever EA por diretrizes nacionais e internacionais de prevenção e reabilitação cardiovascular.2Nesse sentido, métodos alternativos ao TCPE têm sido utilizados, como equações para predizer a FC máxima e escalas relacionadas à percepção subjetiva de esforço.16 No entanto, estudos utilizando TF para prescrever EA são escassos, mesmo em pessoas saudáveis.O TF pode ser aplicado em cicloergômetro ou esteira, comumente de forma semelhante ao protocolo do TCPE. Ao final de cada estágio, o paciente tem seu conforto de fala questionado, geralmente lendo um parágrafo padrão e respondendo à pergunta “Está confortável para falar?”. Existem 3 alternativas de resposta: “sim”, a fala continua confortável durante o exercício ; “talvez”, o paciente percebe desconforto na fala ; e “não”, o paciente não consegue ler o parágrafo confortavelmente . O último estágio (TF−) é considerado um critério para interrupção do teste.Não se sabe se a FC alcançada nos estágios do TF é uma medida válida para prescrever a intensidade do EA e se esses estágios podem ser comparados com a FC no pico do TC6min e nos LVs do TCPE. Acreditamos que a FC nos estágios do TF esteja associada ao padrão de referência, aos valores dos LVs, bem como com a frequência cardíaca pico (FCpico) alcançada em um TC6min. Compreender as semelhanças e as associações entre essas 3 diferentes ferramentas de prescrição pode facilitar a prática clínica, fornecendo informações sobre uma ferramenta adicional para prescrever EA, que pode fornecer um parâmetro de intensidade independente, individualizado e confiável. Portanto, objetivamos analisar e comparar a FC nos estágios do TF e na FCpico no TC6min, bem como compará-las com a FC nos LVs do TCPE.O presente estudo foi aprovado pelo Comitê de Ética em Pesquisa com Seres Humanos da Universidade do Estado de Santa Catarina (número 96032818.4.0000.0118) e conduzido de acordo com a Resolução 466/12 do Conselho Nacional de Saúde e a Declaração de Helsinque. Todos os pacientes assinaram o termo de consentimento livre e esclarecido.Uma amostra intencional e não probabilística de pacientes de ambos os sexos, com idade entre 40 e 80 anos, diagnosticados com doença cardiovascular crônica clinicamente estável  foi recrutada no Núcleo de Cardio-oncologia e Medicina do Exercício da Universidade do Estado de Santa Catarina.Os critérios de inclusão foram alfabetização para compreender suficientemente o TF e assinar o termo de consentimento livre e esclarecido. Os critérios de exclusão foram alterações de fala ou musculoesqueléticas, acuidade visual comprometida que impedisse o paciente de ler o parágrafo, dor intensa inespecífica ou angina durante os testes, doença neurológica diagnosticada por meio de teste cognitivo ou doenças respiratórias.Os dados foram coletados em 3 dias. O primeiro dia consistiu em uma entrevista estruturada e medidas antropométricas, incluindo índice de massa corporal, avaliação cognitiva  e avaliação do TCPE. No segundo dia, foram realizados dois TC6min com intervalo de 30 minutos entre eles. O TF foi realizado no terceiro dia. Todos os pacientes foram avaliados dentro de 48 e 72 horas entre os testes e no mesmo período do dia.17O mini exame do estado mental foi utilizado como critério de inclusão. Este exame foi aplicado com uma pontuação de 0 a 30 com pontos de corte de acordo com o nível de educação: analfabeto (20 pontos), 1 a 4 anos de estudo (25 pontos), 5 a 8 anos de estudo , 9 a 11 anos de estudo (28 pontos) e > 11 anos de estudo (29 pontos).O TCPE foi realizado em esteira  utilizando um protocolo de rampa individualizado. A velocidade e a inclinação variaram a cada minuto para permitir que o teste durasse entre 8 e 12 minutos. Os pacientes realizaram recuperação ativa após o protocolo .2-PetCO2 .Utilizamos um circuito aberto computadorizado  para analisar os gases expiratórios e a ventilação, enquanto um frequencímetro de pulso  avaliou a FC durante o teste. O primeiro (LV1) e o segundo (LV2) LV foram identificados usando os métodos V-slope e PetOPara a segurança dos pacientes, foram adotados os seguintes critérios de interrupção do teste: sinais ou sintomas de intolerância ao exercício , resposta cardiovascular inadequada , ou solicitação de interrupção pelo participante.18 Duas repetições do TC6min foram realizadas em uma pista de 30 metros de extensão, e os pacientes foram solicitados a caminhar a maior distância possível em 6 minutos.18O TC6min foi aplicado de acordo com as diretrizes atuais.19 foram avaliados antes, imediatamente após e 2 minutos após os testes. A FC e a saturação de pulso de oxigênio também foram avaliadas a cada minuto durante os testes.18 Para análise, utilizamos a FCpico apresentada durante o teste com a maior distância percorrida. Os critérios para interrupção do TC6min foram semelhantes aos do TCPE.A FC , a pressão arterial , a saturação de pulso de oxigênio  e o esforço percebido O TF foi realizado em esteira  usando um protocolo incremental independente e individualizado de acordo com a distância predita do TC6min. A FC, pressão arterial, saturação de pulso de oxigênio e esforço percebido foram avaliados antes, imediatamente após e 2 minutos após o TF. Também monitoramos a FC e a saturação de pulso de oxigênio durante o teste, enquanto o esforço percebido foi questionado apenas ao final de cada estágio.A carga foi aumentada a cada 2 minutos durante o teste (estágios). Nos últimos 30 segundos de cada estágio, solicitamos aos pacientes que recitassem o seguinte parágrafo de 36 palavras:“Saúde é um estado de completo bem-estar físico, mental e social, e não consiste apenas na ausência de doença ou de enfermidade. É um direito fundamental, que deve ser assegurado sem distinção de raça, religião ou condição social.”Logo após a leitura, foi perguntado aos pacientes “Está confortável para falar?”, e as respostas possíveis eram “sim” (TF+); “mais ou menos” (TF±); ou “não” (TF−). O TF− foi considerado um critério para interromper o TF. Outros critérios para interrupção do teste foram semelhantes ao TCPE e TC6min.20Um protocolo incremental contínuo foi conduzido com incrementos de velocidade ou inclinação no início de cada estágio. Foi usada uma equação para calcular a distância predita do TC6min (TC6minpred).Figura 1S, Material Suplementar).Conhecendo o TC6minpred, a velocidade média foi estimada da forma seguinte:Os dados foram analisados usando o software Statistical Package for the Social Sciences, versão 20.0 . As variáveis contínuas foram descritas como média e desvio padrão ou mediana e intervalo interquartílico, de acordo com a normalidade dos dados. A normalidade da distribuição dos dados foi verificada com o teste de Shapiro-Wilk.As variáveis com distribuição normal foram comparadas por meio da ANOVA de medidas repetidas, seguida do post-hoc de Bonferroni, enquanto o teste de Friedman foi usado para variáveis com distribuição não normal. O coeficiente de correlação de Pearson ou Spearman avaliou a associação entre as variáveis. O nível de significância adotado na análise estatística foi de 5%.O tamanho da amostra foi calculado usando o GPower 3.1 em um estudo piloto com 5 participantes, considerando nível de significância bidirecional (α) de 5% e poder de 80% . Com base na correlação entre FC durante os estágios do TF (TF+ e TF±) e LV1  e FCpico no TC6min , o tamanho amostral mínimo estimado foi de 20 pacientes.Avaliamos 30 pacientes, dos quais 8 foram excluídos devido a alterações musculoesqueléticas (n = 3), erro de medida (n = 3), angina (n = 1) e dor periumbilical (n = 1). Dos 22 pacientes incluídos, 13 eram do sexo masculino  .Conforme demonstrado na A Uma correlação moderada foi verificada entre TF+ e LV1. No entanto, a FC no TF± não se correlacionou com a FC no LV e a FCpico no TC6min. A FC no TF− foi fortemente correlacionada com o LV2. A FCpico no TC6min não foi correlacionada com a FC no LV1, mas sim com a FC no VT2 e TF−. A Este é o primeiro estudo que compara a FC avaliada nos LVs do TCPE com a FC nos estágios de um protocolo de TF independente e individualizado e durante um teste de campo (TC6min). A FC no TF+ e TF± foi semelhante à FC no LV1, enquanto a FC no TF− e a FCpico no TC6min foram semelhantes à FC no LV2. A FC no TF+ e TF− se correlacionou com a FC no VT1 e VT2, respectivamente. Não foi encontrada correlação entre a FCpico no TC6min e a FC nos estágios do TF.4 Se um TCPE não estiver disponível, o teste ergométrico pode ser usado para obter parâmetros cardiovasculares máximos. Na ausência desses testes, a avaliação funcional e a prescrição de EA geralmente são realizadas com base em um teste de campo, como o TC6min e o TF.8 Embora os estágios do TF estejam fisiologicamente relacionadas aos LVs, seu uso para prescrição de EA ainda é escasso, principalmente para pacientes cardíacos.Os métodos individualizados de prescrição de EA mais reconhecidos são o consumo máximo de oxigênio e os LVs identificados pelo TCPE.16 Apesar de utilizarem métodos diferentes, estudos demonstraram semelhanças entre a FC e o consumo de oxigênio nos estágios do TF e LV em pacientes com doenças cardíacas, sugerindo que os estágios do TF podem ser usadas para prescrever EA.22No presente estudo, investigamos um protocolo de TF e encontramos correlações entre a FC no LV1 e TF+ e TF±. O TF não possui um protocolo padronizado e pode ser realizado com diferentes equipamentos, progressões de carga e durações dos estágios, e a fala pode ser desafiada usando parágrafos ou contagem.22 Além disso, foi encontrada uma forte correlação entre a FC no TF− e LV2. Embora haja escassez de estudos comparando a FC entre esses dois momentos em pacientes com doença cardiovascular, associações entre o consumo de oxigênio e a FC no TF− e LV2 foram encontradas em indivíduos saudáveis e atletas,24 sugerindo, portanto, que o TF− pode orientar a prescrição de EA e refletir a prescrição com base no LV2.Nossos resultados foram semelhantes aos de Brawer et al. (2006), que aplicaram o TF em pacientes com doença arterial coronariana estável e demonstraram nenhuma diferença na estimativa do LV1 e na viabilidade da prescrição de EA pela FC de acordo com o TF.25Outro método comumente utilizado em programas de reabilitação cardiovascular para prescrição da intensidade de EA é baseado no TC6min. Gremeaux et al. (2011) compararam os efeitos de 3 prescrições individualizadas de treinamento físico em indivíduos treinando em intensidade moderada. Encontraram valores semelhantes entre a FCpico no TC6min e a FC alvo recomendada. Outro estudo de Calegari et al. compararam a FC no LV1 durante os últimos 30 segundos do TC6min em pacientes com doença arterial coronariana em tratamento com betabloqueadores. Os autores encontraram concordância entre a FC avaliada ao final do TC6min e o LV1, sugerindo que a FC ao final do TC6min foi adequada para prescrever e monitorar o EA nessa população.1 nossos dados sugerem que a FCpico no TC6min pode não ser adequada para prescrever o limite inferior de intensidade de EA em pacientes com doença cardiovascular crônica. Além disso, a FCpico no TC6min, como LV2 e TF−, pode representar o limite superior da prescrição de EA.Esses achados corroboram a ideia de utilizar a FC no TC6min como método de prescrição da intensidade do exercício, a FC medida no TC6min sendo semelhante à FC avaliada no LV1. No entanto, encontramos diferenças entre a FCpico no TC6min e a FC no LV1 em nosso estudo, enquanto a FCpico no TC6min foi semelhante à FC no LV2 e no TF−. De acordo com as recomendações mais recentes, a prescrição de EA deve ser realizada entre o primeiro e o segundo VT;Essa semelhança nunca foi descrita na literatura e alguns aspectos do presente estudo podem ter influenciado esse resultado. Os pacientes faziam parte de um programa de reabilitação cardiovascular que realiza regularmente o TC6min. Além disso, em relação à distância percorrida, nossa amostra atingiu uma distância percorrida acima de 100% do previsto, demonstrando pouco comprometimento da capacidade funcional. Além disso, o TC6min foi realizado em campo aberto, enquanto o TCPE e o TF foram realizados em ambientes controlados. Adicionalmente, estudos anteriores utilizaram a FC do minuto final do TC6min em vez da FCpico durante o teste, que foi utilizada em nossas análises.18 Por outro lado, o TF requer menos espaço físico, pode ser realizado em cicloergômetro ou esteira, segue um protocolo de rampa incremental semelhante ao TCPE, e é de fácil aplicação e equivalente ao padrão de referência.Embora comumente utilizado na reabilitação cardiovascular, o TC6min requer espaço físico devido à pista de 30 metros de extensão. Além disso, o teste é auto-cadenciado e apresenta variações inter e intraindividuais de velocidade e desempenho.O presente estudo não está isento de limitações. Os pacientes faziam parte de um programa de reabilitação cardiovascular fase III e possivelmente apresentavam melhores condições físicas do que os pacientes nas fases iniciais da reabilitação. Além disso, a percepção da fala confortável é subjetiva e foram perdidos alguns dados devido a erro de mensuração.As comparações entre o TF e o TCPE foram semelhantes à literatura, exceto entre o TF e TC6min. Acreditamos que o TF apresente parâmetros associados a variáveis fisiológicas e do TCPE . Nossos resultados também mostraram a aplicabilidade clínica do TF como uma ferramenta fácil, segura e individualizada para a prescrição de EA.De acordo com o protocolo proposto, a FC no TF+ e TF− reflete a FC no LV1 e LV2, respectivamente, demonstrando que o TF é um teste objetivo e de baixo custo para auxiliar a prescrição de EA em pacientes com doenças cardiovasculares crônicas. Por outro lado, a prescrição de EA utilizando a FCpico no TC6min deve ser realizada com cautela, pois alguns estudos mostraram sua correlação com o LV1, diferentemente do que foi verificado em nosso estudo, no qual a FCpico no TC6min foi semelhante à FC no LV2 e TF−. Sugerimos futuros ensaios clínicos randomizados usando parâmetros do TF para prescrever EA em pacientes com doenças cardiovasculares. 1 The general recommendation for the practice of physical exercise, according to the Brazilian Society of Cardiology, is 150 minutes per week divided into 3 to 5 weekly sessions. The appropriate practice of physical exercise aims to reduce blood pressure, risk of cardiovascular events and risk of mortality in cardiovascular patients.2The main goal of cardiovascular rehabilitation is to improve cardiorespiratory fitness using aerobic exercise (AE).1 CPET analysis allows identification of the most adequate and recommended variables, such as maximal oxygen uptake, workload, maximal heart rate (HR), HR reserve, and ventilatory thresholds (VTs).5 However, this test still has limited access, especially in developing countries, due to high costs, the need for highly trained professionals, and complex analyses.5The cardiopulmonary exercise test (CPET) is the reference test to assess cardiorespiratory function and prescribe AE intensity.6 Another method uses absolute or relative HR values achieved during submaximal tests, such as the 6-minute walk test (6MWT)9 and the talk test (TT).11 The TT is a validated and accessible test, based on an incremental load protocol, and it uses the perception of speech comfort as a marker for exercise intensity.13 Recently, the TT has been recommended to assess functional capacity and prescribe AE by national and international cardiovascular prevention and rehabilitation guidelines.2In this sense, alternative methods to CPET have been used, such as equations to predict the maximal HR and scales related to subjective perceived exertion.16 Nevertheless, studies using TT to prescribe AE are scarce, even in healthy people.The TT can be applied using a cycle ergometer or treadmill, commonly in a similar way to the CPET protocol. At the end of each stage, patients have their speech comfort challenged, usually reading a standard paragraph, and answering the question “Can you speak comfortably?”. There are 3 answer alternatives: “yes,” the speech is still comfortable during exercise ; “maybe,” the patient notices discomfort in the speech ; and “no,” the patient is unable to read the paragraph comfortably . The last stage (TT−) is considered a criterion for interrupting the test.It is unknown whether HR achieved at TT stages is a valid measure to prescribe the AE intensity and whether these stages can be compared with the HR at the peak of the 6MWT and at the VTs from a CPET. We believe that the HR at the TT stages is associated with the reference standard, the values on the VTs, as well as with the peak heart rate (HRpeak) achieved in a 6MWT. Understanding the similarities and associations between these 3 different prescription tools can facilitate clinical practice by providing information about an additional tool to prescribe AE, which can provide an independent, individualized, and reliable intensity parameter. Therefore, we aimed to analyze and compare the HR at the TT stages and at the HRpeak in the 6MWT, as well as to compare them with HR at VTs of CPET.This study was approved by the Human Research Ethics Committee (number 96032818.4.0000.0118) and conducted according to Resolution 466/12 of the National Health Council and the Declaration of Helsinki. All patients signed the informed consent form.An intentional and non-probability sample of patients of both sexes, aged between 40 and 80 years, diagnosed with clinically stable chronic cardiovascular disease  was recruited in the Center of Cardio-Oncology and Exercise Medicine of Santa Catarina State UniversityInclusion criteria were sufficient literacy to understand the TT and sign the informed consent form. Exclusion criteria were speech or musculoskeletal alterations, impaired visual acuity that prevented the patient from reading the paragraph, non-specific intense pain or angina during the tests, neurological disease diagnosed using a cognitive test, or respiratory diseases.Data were collected in 3 days. The first day consisted of a structured interview and anthropometric measurements, including body mass index, cognitive assessment , and CPET assessment. On the second day, two 6MWT were performed with a 30-minute interval in between. The TT was performed on the third day. All patients were evaluated within 48 and 72 hours between the tests at the same period of the day.17The mini-mental state exam was used as an inclusion criterion. This exam was applied using a score from 0 to 30 with cut-off points according to educational level as follows: illiterate (20 points), 1 to 4 years of schooling (25 points), 5 to 8 years of schooling (26.5 points), 9 to 11 years of schooling (28 points), and > 11 years of schooling (29 points).The CPET was performed on a treadmill  using an individualized ramp protocol. Speed and inclination varied each minute to enable the test to last between 8 and 12 minutes. Patients performed an active recovery after the protocol .2-PetCO2 methods .We used a computerized open circuit  to analyze expiratory gases and ventilation, while a pulse rate monitor  assessed HR during the test. The first (VT1) and second (VT2) VT were identified using V-slope and PetOThe following criteria for interrupting the test were adopted for the safety of patients: signs or symptoms of exercise intolerance , inadequate cardiovascular response (blood pressure and/or HR), or if requested by the participant.18 Two repetitions of the 6MWT were performed on a 30-meter-long track, and patients were requested to walk the longest distance possible in 6 minutes.18The 6MWT was applied according to current guidelines.19 were assessed before, immediately after, and 2 minutes after the tests. HR and pulse oxygen saturation were also assessed each minute during the tests.18 For analysis we used the HRpeak presented during the test with the higher walked distance. Criteria for interrupting the 6MWT were similar to CPET.HR , blood pressure , pulse oxygen saturation , and perceived exertion The TT was performed on a treadmill  using an independent and individualized incremental protocol according to the predicted 6MWT distance. HR, blood pressure, pulse oxygen saturation, and perceived exertion were assessed before, immediately after, and 2 minutes after the TT. We also monitored HR and pulse oxygen saturation during the test, while perceived exertion was questioned only at the end of each stage.The load was increased every 2 minutes during the test (stages). In the last 30 seconds of each stage, we requested patients to recite the following paragraph of 36 words:“Health is a state of complete physical, mental, and social well-being, not only the absence of disease or illness. It is a fundamental right that must be ensured without distinction of race, religion, or social condition.”Right after reading, the patients were asked “Can you speak comfortably?”, and answers could be “yes” (TT+); “uncertain” (TT±); or “no” (TT−). The TT− was considered a criterion to interrupt the TT. Other criteria for interrupting the test were similar to the CPET and 6MWT.20A continuous incremental protocol was conducted with speed or incline increments at the beginning of each stage. An equation was used to calculate the predicted 6MWT distance (6MWDpred).Figure 1S, Supplementary Material).Knowing the 6MWDpred, mean speed was estimated as follows: Data were analyzed using the Statistical Package for the Social Sciences software, version 20.0 . Continuous variables were described as mean and standard deviation or median and interquartile range, according to data normality. The normality of the data distribution was tested with the Shapiro-Wilk test.Variables with normal distribution were compared using the repeated measures ANOVA, followed by Bonferroni’s post-hoc, while the Friedman’s tests was used for variables with non-normal distribution. Pearson’s or Spearman’s correlation coefficient assessed association between variables. The significance level adopted in the statistical analysis was 5%.The sample size was calculated using GPower 3.1 in a pilot study with 5 participants, considering bidirectional significance level (α) of 5% and power of 80% (β = 0.20). Based on the correlation between HR during TT stages (TT+ and TT±) and VT1  and HRpeak at 6MWT , the minimal estimated sample size was 20 patients.We evaluated 30 patients, and 8 of them were excluded due to musculoskeletal alterations (n = 3), measurement error (n = 3), angina (n = 1), and periumbilical pain (n = 1). Of the 22 included patients, 13 were males (59.1%) .As shown in A moderate correlation was found between TT+ and VT1. Nonetheless, HR at TT± was not correlated with HR at VT, and HRpeak in the 6MWT. HR at TT− was strongly correlated with VT2. HRpeak in the 6MWT did not correlate with HR at VT1 but correlated with HR at VT2 and TT−. This is the first study comparing the HR assessed at VTs of the CPET with the HR at stages of an independent and individualized TT protocol, and during a field test (6MWT). HR at TT+ and TT± were similar to HR at VT1, while HR at TT− and HRpeak in the 6MWT were similar to HR at VT2. HR at TT+ and TT− correlated with HR at VT1 and VT2, respectively. No correlation was found between HRpeak in the 6MWT and HR at TT stages.4 If a CPET is unavailable, graded exercise test may be used to obtain maximal cardiovascular parameters. In the absence of these tests, a functional assessment and AE prescription are usually carried out based on a field test, such as 6MWT and TT.8 Although the TT stages are physiologically related to the VTs, their use for AE prescription is still scarce, mainly for cardiac patients.The most recognized individualized AE prescription methods are the maximal oxygen uptake and VTs identified using the CPET.16 Despite using different methods, studies have demonstrated similarities between HR and oxygen uptake at TT stages and VTs in patients with cardiac disease, suggesting that TT stages may be used to prescribe AE.22In the present study, we explored a TT protocol and found correlations between HR at VT1 and TT+ and TT±. The TT does not have a standardized protocol and may be performed with different equipment, load progressions, and stage lengths, and the speech can be challenged using paragraphs or counting.22 In addition, a strong correlation was found between HR at TT− and VT2. Although there is a lack of studies comparing HR between these two moments in patients with cardiovascular disease, associations between oxygen uptake and HR at TT− and VT2 were found in healthy individuals and athletes,24 therefore suggesting that TT− may guide AE prescription and reflect the prescription based on VT2.Our results were similar to Brawer et al. (2006), who applied the TT in patients with stable coronary artery disease and demonstrated no difference in estimating VT1 and the viability of prescribing AE using HR according to TT.25Another method commonly used at cardiovascular rehabilitation programs for prescribing AE intensity is based on the 6MWT. Gremeaux et al. (2011) compared the effects of 3 individualized exercise training prescriptions in individuals training at a moderate intensity. They found similar values between HRpeak in the 6MWT and the recommended target HR. Another study by Calegari et al. (2021) compared HR at VT1 during the last 30 seconds of the 6MWT in patients with coronary artery disease on β-blockers treatment. The authors found an agreement between HR assessed at the end of the 6MWT and VT1, suggesting that HR at the end of the 6MWT was adequate to prescribe and monitor AE in this population.1 our data suggest that HRpeak in the 6MWT could not be adequate to prescribe lower limit AE intensity in patients with chronic cardiovascular disease. Furthermore, the HRpeak at 6MWT, such as VT2 and TT−, may represent the upper limit of AE prescription.These findings support the idea of using the HR at 6MWT as an exercise intensity prescription method, with HR measured in the 6MWT being similar to HR assessed at VT1. However, we found differences between HRpeak in the 6MWT and HR at VT1 in our study, while HRpeak in the 6MWT was similar to HR at VT2 and at TT−. According to the most recent recommendations, AE prescription should be performed between the first and second VTs;This similarity has never been described in the literature, and some aspects of the present study may have influenced this outcome. Patients were part of a cardiovascular rehabilitation program that regularly performs the 6MWT. In addition, regarding the walked distance, our sample reached a distance walked above 100% of the predicted, demonstrating little impairment of functional capacity. Moreover, the 6MWT was performed in an open field, whereas CPET and TT were performed in controlled environments. In addition, previous studies used HR from the final minute of the 6MWT instead of HRpeak during the test, which was used in our analyses.18 On the other hand, the TT requires less physical space, may be performed in a cycle ergometer or treadmill, follows an incremental ramp protocol similar to CPET, and is easy to apply and equivalent to the reference standard.Although commonly used in cardiovascular rehabilitation, the 6MWT requires physical space because of the 30-meter-long track. Furthermore, the test is self-paced and presents inter- and intra-individual variations of speed and performance.This study is not free of limitations. Patients were part of a phase III cardiovascular rehabilitation program and possibly presented better physical conditions than patients in the earlier phases of rehabilitation. Moreover, the perception of comfort speech is subjective, and data were lost due to measurement error.Comparisons between TT and CPET were similar to the literature, except between TT and 6MWT. We believe that TT presents parameters associated with physiological and CPET variables . Our results also showed the clinical applicability of TT as an easy, safe, and individualized tool for AE prescription.According to the proposed protocol, HR at TT+ and TT− reflect HR at VT1 and VT2, respectively, demonstrating that TT is an objective and low-cost test to assist AE prescription in patients with chronic cardiovascular conditions. In contrast, AE prescription using HRpeak in the 6MWT must be carried out with caution since some studies have shown its correlation with VT1, differently from what was found in our study, in which HRpeak in the 6MWT was similar to HR at VT2 and TT−. We suggest further randomized clinical trials using TT parameters to prescribe AE in patients with cardiovascular diseases.


All analyzed CSF biomarkers, except neurofilament light chain and Aβ1/x–42, were increased several-fold. In blood, none of these biomarkers were significantly different between the Skogholt and control groups. MRI volumetric segmentation revealed decreases in the ventricular, white matter, and choroid plexus volumes in the Skogholt group, with an accompanying increase in white matter lesions. The cortical thickness and subcortical gray matter volumes were increased in the Skogholt group. Pathophysiological changes resulting from choroidal dysfunction and/or abnormal CSF turnover, which may cause the increases in CSF protein and brain biomarker levels, are discussed.Skogholt’s disease is a rare neurological disorder that is only observed in a small Norwegian kindred. It typically manifests in adulthood with uncharacteristic neurological symptoms from both the peripheral and central nervous systems. The etiology of the observed cerebral white matter lesions and peripheral myelin pathology is unclear. Increased cerebrospinal fluid (CSF) concentrations of protein have been confirmed, and recently, very high concentrations of CSF total and phosphorylated tau have been detected in Skogholt patients. The symptoms and observed biomarker changes in Skogholt’s disease are largely nonspecific, and further studies are necessary to elucidate the disease mechanisms. Here, we report the results of neurochemical analyses of plasma and CSF, as well as results from the morphometric segmentation of cerebral magnetic resonance imaging. We analyzed the biomarkers Aβ Skogholt’s disease is a rare, slowly progressing, central and peripheral neurodegenerative disorder only observed with maternal inheritance in a small Norwegian family line. So far, it has been diagnosed in four generations in a community in the southeastern part of Norway, and it was named after the local community physician who first described it ,2. MinorThey found greatly elevated cerebrospinal fluid (CSF) concentrations of total protein and extensive cerebral white matter lesions, together with signs of peripheral nervous system involvement, originally interpreted as a combined central and peripheral demyelinating disorder . In thre42) [Later findings included greatly increased concentrations in the CSF of copper (Cu), iron (Fe), total tau (t-tau), and phosphorylated tau (p-tau), combined with normal to low levels of amyloid beta 42 protein (Aβ42) ,4, but n42) . Subjectively experienced cognitive difficulties are common, and a cognitive screening battery disclosed a significantly prolonged Trail-Making-Test-B time, suggesting some difficulty in executive functioning .The cause of the strikingly unusual combination of clinical and paraclinical findings in Skogholt’s disease is unknown but likely represents a genetically linked neurodegenerative condition not described outside the affected family line ,2,3,4.x–42, Aβx–4o, and Aβx–38; phosphorylated tau (p-tau); and total tau (t-tau), as well as the astroglia-associated glial fibrillary acidic protein (GFAP), the axonal biomarker neurofilament light chain (NfL), the microvascular marker platelet-derived growth factor receptor beta (PDGFRβ), and beta-trace protein (βTP). In addition, we obtained segmental data of the cortical thicknesses and volumes of brain substructures using algorithmic processing of brain MRI data. In the present study, our objective was to further study the pathophysiology of Skogholt’s disease, as well as to disclose the characteristics of this disease. We applied a comprehensive battery of markers in CSF and blood, including the core markers of Alzheimer’s disease (AD), specifically the amyloid-β species AβWe included three separate groups: Skogholt cases, lab controls, and MRI controls. Belonging to the affected family line was a prerequisite to consider a diagnosis of Skogholt’s disease. An additional increase in CSF total protein above 1 g/L  was considered sufficient for the diagnosis. Increased CSF total protein in the range between 0.45 and 1.0 g/L was also considered sufficient for a definite diagnosis if accompanied by clinical symptoms or MRI findings consistent with Skogholt’s disease.Eleven Skogholt patients that were capable of informed consent and physically fit enough to attend were included in the study. All cases belonged to one kindred from a community in the southeastern inland part of Norway. Three additional cases were unable to participate due to age-related frailty or concurrent morbidity at the time of inclusion.The lab control group consisted of 14 individuals recruited from the Department of Neurology at the Innlandet Hospital Trust in Lillehammer. Patients not expected to receive an inflammatory or neurodegenerative diagnosis were targeted for inclusion, while patients with certain multiple sclerosis or known dementia were not considered eligible. The lab control group was thus heterogeneous, as previously described . Samples42/40 ratio ≤ 0.077), with normal results on a cognitive screening battery, and without subjective cognitive complaints. Two eligible controls were excluded due to failed algorithmic parcellation of MRI data.For the comparison of cortical and parenchymal segmentation data, we included 60 MRI controls from the Dementia Disease Initiation (DDI) project, which has more than 500 participants [The study protocol was approved by the Regional Committee for Medical and Health Research Ethics, South-East Region, Norway, Ref. No. 556-04224 and No. 2013/1017. The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients before enrollment.Samples of plasma and CSF were obtained and prepared as described previously . Before 1–42 and Aβ1–40 concentrations were measured using a fully automated Lumipulse instrument  as described previously [x–38, Aβx–40, and Aβx–42 were measured using a MesoScale Discovery triplex assay. CSF NfL and GFAP concentrations were measured using in-house ELISAs as previously described [From CSF and EDTA-plasma samples obtained previously , we analeviously . The Aβ escribed ,8,9. SolThe beta-trace protein (prostaglandin D synthase) concentration was measured via nephelometry on an Atellica NEPH 630 System . The measurements were performed by board-certified laboratory technicians who were blinded to the clinical data.Cerebral MRI of Skogholt patients was obtained on a Philips Achieva MRI system with a magnetic field strength of 1.5 Tesla. The MRI protocol included 3D FLAIR, axial T2, diffusion, inflow angio, and SWI as well as 3DT1 scanning before and after contrast enhancement with a macrocyclic gadolinium contrast agent (gadoteric acid). MRI from the DDI controls used in the current study were obtained on eight scanners distributed in six centers. The scanner systems, sequence parameters, and number of subjects are detailed in Cortical reconstruction and volumetric segmentation were performed with FastSurfer ,11. ThisWhite matter hyperintensities  (WMHs) wAll statistical computing and data visualizations were performed in RStudio using R version 4.1.3   with extDue to the small numbers of cases and controls, judgements regarding the distribution of data were unreliable. Therefore, we used both Student’s two-tailed two-sample t-test and the Wilcoxon rank-sum test, i.e., the Mann–Whitney U-test, with α pragmatically set to 0.01 when judging group differences and correspondingly provide the raw data of descriptive statistics for each group using the mean and median with the standard deviation (SD) and interquartile, i.e., 1st to 3rd quartile, range (IQR). Group differences in standardized measurements or standardized ratios (measurements with ICV for MRI volume markers) are explored in graphs showing the standardized linear regression coefficients  of group status for each marker.The adjustment of coefficients was carried out by including appropriate covariates in the regression models. Continuous variables such as age and ICV were standardized, while dichotomous variables, e.g., sex or MRI magnetic field strength (1.5 or 3T), were kept unchanged as binary variables.To verify the confidence intervals, they are also shown as the 2.5th to 97.5th percentile ranges of the bootstrap distributions of the estimates/coefficients based on 20,000 resampled datapoints in the dataset stratified by group and gender.The Demographics of the Skogholt cases and the two control groups are given in The opening pressure upon lumbar puncture was normal in all Skogholt patients, ranging from 10.5 to 19.5 cm water, with a mean of 14.4 cm, which was not significantly different from the controls.t-test and Mann–Whitney U-test), except for Aβ1–42, Aβx–42, and NfL, as shown in 1–42, Aβx–42, and NfL were borderline significantly increased according to the U-test but not the t-test).The measured CSF biomarkers were significantly increased in the Skogholt group   .The majority of the examined Skogholt cases had grade-three Fazekas, i.e., confluent deep white matter T2 hyperintensities on cerebral MRI , while mThe intracranial volume (ICV) was significantly smaller in the Skogholt group, even when adjusting for age, sex, and magnetic field strength . Only a An algorithmic evaluation of the MRI scans showed generally increased cortical thickness in the Skogholt group, even when adjusting for age, gender, magnetic field strength, and ICV . The corRelative to the ICV  and compared to healthy controls from the DDI project, the Skogholt cases had reduced total white matter, CSF, and choroid plexus volumes and increased white matter changes . The volSummary statistics of the raw data from MRI parcellation are provided in Increased CSF total protein concentrations, extensive white matter hyperintensities, and pathological CSF biomarkers of neurodegeneration are established characteristics of Skogholt’s disease ,3,4.The present work adds to the literature on Skogholt’s disease with an expanded set of CSF and plasma biomarkers and with MRI morphometry. MRI data revealed strikingly decreased white matter volumes, decreased choroid plexus volumes, increased gray matter volumes, and increased cortical thickness compared to the control group. The extended biomarker panel confirmed previous findings of increased CSF T-tau and P-tau  and reve42 in Skogholt’s disease [x–40 and Aβx–38 found in Skogholt CSF in this study. The ELISA technique (Innotest kit) previously used to measure the Aβ42 concentration [40 or Aβ38 [1–40 and Aβ1–42 and a triplex method for Aβx–38, Aβx–40, and Aβx–42.Although the previously reported low levels of CSF Aβ disease  appear t or Aβ38 . The curThe CSF beta-trace protein (βTP) was increased almost seven-fold, substantially more than what can be accounted for by the, on average, 3.5-fold increase in CSF total protein previously reported . However38, Aβ40, NfL, and GFAP) up to 8–10-fold (for t-tau and p-tau). The lack of brain atrophy on MRI indicates that these increases are not related to neurodegeneration. A possible explanation is deranged CSF production affecting the dilution and turnover of constituents, which would allow for a higher CSF concentration of brain proteins. Future research should specifically determine the CSF production and clearance rates in Skogholt’s disease. With MRI, we find that the choroid plexus is smaller in Skogholt’s disease compared to controls, a possible clue to the primary pathology of the condition. CSF protein concentrations are traditionally used as a surrogate marker for the blood–brain barrier (BBB) as well as blood–nerve barrier (BNB) integrity. In chronic inflammatory polyneuropathy, which appears to exist in some patients with Skogholt’s disease [High CSF protein concentrations have previously been reported in conditions with slow CSF turnover ,27. It i disease . BNB dam disease . Based oThe MRI findings in the Skogholt cases were unexpected. Compared to the controls, we report decreased white matter and increased gray matter volumes. Additionally, we report decreased choroid plexus and ventricle volumes. The decreased white matter volume may be explained by demyelination or degeneration secondary to the pathological composition of the CSF . PreviouIncreased gray matter volumes may be explained by swelling or alternatively by compensatory mechanisms, e.g., an increased number of local interneuronal connections and dysmyelination. Dysmyelination and BBB integrity in Skogholt’s disease are topics for future studies.The scope of the current study is limited by its small numbers of cases and controls available for fluid marker comparison. The lab control group was diverse and was not sampled as early in the morning as was possible for the Skogholt group. The MRI control group was sufficiently large, but lab results were not available for comparison. Furthermore, the cases and the MRI controls were not examined using the same MRI scanner, introducing the possibility for biased scanner results. However, the morphometric differences were consistent for the different MRI systems, and we corrected for field strength. Additionally, if there were differences due to scanner effects, we would not expect to see the present pattern of group differences. Morphometry on 3T systems is known to report increased cortical thickness compared to 1.5 T systems , the oppThe extraordinarily high CSF biomarker levels in Skogholt’s disease are remarkable but must be considered together with the overall increase in CSF proteins, which are altogether more compatible with disturbed CSF dynamics than neurodegeneration. MRI findings of confluent white matter lesions are typical but not necessary to establish a diagnosis of Skogholt’s disease. Our algorithmic processing of MRI data showed decreases in white matter and choroid plexus volumes accompanied by increases in cortical thickness and volume, as well as an increased volume of subcortical gray matter.Future studies on Skogholt’s disease will benefit from including more cases and avoiding systematic biases when the laboratory analysis and neuroimaging are not distributed over several locations.