Brain disorders in euthyroid Hashimoto’s thyroiditis patients

Hashimoto’s thyroiditis (HT) is an autoimmune disorder characterized by the destruction of thyroid follicular cells by thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TgAb), leading to hypothyroidism. Hashimoto’s encephalopathy (HE) is associated with elevated levels of antithyroid antibodies. An important question is whether brain alterations precede the development of HE and are present in euthyroid patients with HT, and what pathomechanisms could be responsible for these changes. A PubMed search was conducted to identify studies addressing this issue. Numerous questionnaire studies confirmed impairments in cognitive functioning, mental and physical health, and overall well-being in euthyroid HT patients. Additionally, some imaging and mouse model studies indicate that euthyroid patients with HT likely have central nervous system alterations. Antibodies may be involved in the development of these changes. Some research suggests the role of TPOAb and TgAb, while other studies highlight the involvement of coexisting antibodies. Determining whether antibodies are assessed in serum or cerebrospinal fluid (CSF) is crucial. Antibody-specific indices (ASIs) can differentiate between antibodies passively diffusing from the serum and brain-derived antibodies, and could serve as biomarkers for brain alterations in HT patients. Much more research is needed to identify reliable biomarkers and treatments that could improve the quality of life for these patients.


Introduction
Hashimoto's thyroiditis (HT) is the most common thyroid disease in iodine-sufficient areas.This autoimmune disorder is caused by the destruction of thyroid follicular cells by thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TgAb).The consequential thyroid infiltration by lymphocytes leads to the destruction of thyrocytes and hypothyroidism [1,2].Symptoms of hypothyroidism in HT patients are well known, but there is little research on euthyroid HT patients.HT patients who develop hypothyroidism are treated with levothyroxine and considered healthy after TSH level normalization.However, it is known that these patients have an increased chance of developing psychiatric diseases such as depression or anxiety disorders [3].
Hashimoto's encephalopathy (HE), also known as steroid-responsive encephalopathy (SREAT) is an encephalopathy without central nervous system infection or tumor.HE presents with elevations in antithyroid antibodies.The prevalence of HE is low (estimated at 2:100,000), but death and status epilepticus have been reported.The first patient with HE was described by Lord Brain of Eynsham in 1966 and since then, more than 200 cases have been reported.HE is responsive to corticosteroid treatment, so it must be considered after excluding other toxic, metabolic, and infectious causes of encephalopathy.Studies of brain magnetic resonance spectroscopy (MRI) and cerebrospinal fluid (CSF) are often normal or non-specific.[4][5][6][7].A review of 251 cases from the literature listed the most common symptoms of HE: encephalitis signs with convulsions, memory impairment, confusion, speech disorder, gait disturbance, progressive memory impairment, isolated psychiatric disorders, and persecutory delusions [8].

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An important question is whether mild brain disorders precede the development of HE.In the literature, there are few studies on brain alterations in euthyroid patients with HT.There is no evidence that HE is a direct consequence of HT.Neverthles, some studies on the etiology of HE could serve as a foundation for identifying these changes in patients with HT.Identifying such associations in the future studies could indicate that HE is a consequence of HT.It is worth noting that even if HE may not necessarily develop in all HT patients, mild brain damage could still occur in some of them.This review aims to gather available PubMed studies on this subject and look for possible pathogenesis of the development of brain disorders, methods of detection, potential biomarkers, and ideas for further research.

Material and methods
This review is based on articles found using PubMed.The inclusion criteria were as follows: studies on the euthyroid state in Hashimoto's thyroiditis, topics related to CNS damage, research on HE patients focusing on the pathomechanism of CNS damage, and articles published after 2000.The key search terms were: Hashimoto's encephalopathy, Hashimoto's thyroiditis, autoimmune thyroiditis, central nervous system, brain alterations, and brain damage.Of the approximately 4000 articles identified, only 29 were relevant to the topic and were subsequently included in the study.

Questionaries studies
Despite levothyroxine treatment and normal thyroid-stimulating hormone (TSH) levels patients with HT exhibited impairments in cognitive functioning, mental and physical health, and general well-being.Hypothyroid patients undergoing levothyroxine treatment scored notably lower in both the mental and physical components of the health-related quality of life (HRQOL) as well as in the Short Form-12 questionnaire (SF-12v1), compared to the control group, even after adjusting for age and BMI (p < 0.001) [9].Scores of global cognitive function (MMSE-Mini mental state examination), conceptual tracking (TMT-Trail Making Test), verbal divergent thinking (like Phonemic fluency test), and anxiety and depression were found to be significantly worse in patients with HT on long-term levothyroxine replacement compared with the control group [10].A study by Saravanan and others confirmed previous studies as they found that patients on thyroxine replacement with normal TSH levels had worse General Health Questionnaire (GHQ-12) and 12-question 'thyroid symptom questionnaire' (TSQ) scores compared to controls [11].Euthyroid HT patients positive for TPOAb showed poorer results in the three symptom Checklist-90-Revised (SCL-90-R) indices: Positive Symptom Distress Index (PSDI), Global Severity Index (GSI), and Positive Symptom Total (PST), as well as with somatization and obsessive-compulsive symptoms after adjustment for age, gender and thyroid function [12].In another study executive function, visual and verbal memory, attention, as well as acoustic working memory were evaluated using established neuropsychological tests, and no significant differences were found between the euthyroid patients with HT and euthyroid patients undergoing hormonal treatment for goitre or after thyroid surgery.However, a significantly higher mean value of TPOAb was found in HT patients with results below the normal range in the d2 attention test compared to those with normal test results.Also, a significantly greater proportion of HT patients had a z-score of less than -1.5 in the d2 test than patients in the control group [13].

The role of thyroid antibodies
One possible explanation for the development of CNS disorders in patients with HT is the impact of antithyroid antibodies in brain tissues.This hypothesis appears to be supported by questionnaire-based research.Specifically, the behavioral problems subscale of the Strengths and Difficulties Questionnaire (SDQ) showed a positive correlation with Higher TPOAb titers, whereas the level of awareness measured by the Mindful Attention Awareness Scale (MAAS) exhibited an inverse correlation with higher TgAb levels.[14].
The resemblance between thyroid and brain tissues could be the underlying cause of these disorders.Studies confirmed that TgAb, TPOAb, and TSH-receptor antibodies (TSH-RAb) may cross-J o u r n a l P r e -p r o o f react with proteins expressed in the central nervous system, which share local homology with the corresponding thyroid autoantigens.Alpha-enolase (ENO1), aldehyde reductase-I (AKR1A) and dimethylargininase-I (DDAHI) are proteins expressed in both central nervous system (CNS) and thyroid, which share local sequence homology with each of the three autoantigens (thyroperoxidase (TPO), thyroglobulin (TG), TSH-receptor (TSH-R)).Autoantibodies against these proteins were found to be increased in the serum and CSF of patients with HE [15,16] (figure 1).It is unknown whether TPOAb and TGAb antibodies may play a role in the development of CNS disorders in patients with HT who are euthyroid and whether they may precede the onset of HE.However, this could be a potential direction for further research in this field.
On the other hand in another case study involving patients with HE, authors found that serum TPOAb did not correlate with cognitive impairment or other neuropsychiatric manifestations.This suggests that TPOAb may not have a pathogenic role in HE [17].Perhaps the problem is in performing the serum test.Testing anti-thyroid antibodies in CSF rather than in serum could enhance diagnostic reliability in HE patients [18].Research by Derch and others appears to support this theory, as they examined 100 patients with unipolar depression for TPOAb and TGAb levels in both serum and CSF.For seropositive samples Antibody-specific indices (ASIs) were calculated.ASIs enable the differentiation between antibodies that passively diffuse from the serum and those derived from the brain.Serum TPOAb were positive in 17 patients, with 13 out of 17 patients showing increased ASIs.TGAb in the serum were detected in three patients, with two of them showing increased ASIs.Intrathecal synthesis of anti-thyroid antibodies and ASIs could be potential biomarkers for brain alterations in HT patients [19] (figure 1).To better understand the pathogenesis of the disease, it would be beneficial to conduct similar studies in patients with Hashimoto's thyroiditis (HT) to investigate the presence of thyroid antibodies in the cerebrospinal fluid of individuals in a euthyroid state.

Possible involvement of other antibodies
Many authors suggest that other coexisting antibodies could be involved in brain alterations associated with HT and even in HT development.Autoantibodies against the α-enolase (ENO1Ab) are considered as a biomarker in HE [20][21][22].There are studies examining this antibody in Hashimoto's disease in mice in a euthyroid state.Lu and others explored whether ENO1Ab was the pathogenic antibody in the thyroid and brain.Two mouse models were used: the mice immunized with TG and the mice immunized with ENO1.In TG-immunized mice, levels of ENO1Ab were significantly increased, while in ENO1-immunized mice TGAb and TSH levels were significantly elevated.The authors suggest that ENO1Ab can cause both thyrocyte damage and brain damage.ENO1Ab may cause thyrocyte damage via antibody-dependent cellular cytotoxicity (ADCC) and initiate or promote the autoimmune inflammatory responses against thyroid tissue.Simultaneously, ENO1Ab may damage brain microvascular endothelial cells through antibody-dependent cell-mediated injury and/or direct cytotoxic activity, which could disrupt the blood-brain barrier, activate the gliocytes, increase intracerebral interleukin 6 (IL-6) expression and Tau phosphorylation.All of this mechanisms contribute to cognitive and behavioral impairments [23] (figure 2).
A very similar study on another protein was conducted by Yang and others.In the protein disulfide-isomerase A3 (PDIA3)-immunized mice model, elevated serum TSH level was found while in classical mouse models of HT increased anti-PDIA3 antibody (PDIA3Ab) were observed.In the thyroid and brain tissues of PDIA3-immunized mice, PDIA3-expressing cells were associated with the formation of the membrane attack complex.PDIA3 protein may serve as a common autoantigen shared by the thyroid and brain tissues, as authors have found it to be involved in both thyroidal and intracerebral damage by activating the complement system.The authors also speculate that the PDIA3 protein and PDIA3 antibodies may play a role in the development of HT.As thyrocyte destruction occurs, intracellular PDIA3-TG adducts form high molecular weight complexes with PDIA3Ab, facilitating the recognition of immature TG by antigen-presenting cells and promoting the development of Hashimoto's thyroiditis.[24] (figure 2).
Additionally, other antibodies may be implicated in CNS damage in patients with HT.In patients with HT Anti-central nervous system auto-antibodies (CNSAbs) were elevated, resulting in impaired myelinogenesis in vitro.Increased production of monocyte-and T-lymphocyte-derived cytokines was observed, which could affect neurotransmitters and diverse brain neurocircuits [25].Two studies identified antibodies in HE patients that could be associated with disease onset and could potentially be detected in HT euthyroid patients.In a case study, authors suggested the possible co-existence of two autoimmune processes: HT and anti-myelin oligodendrocyte glycoprotein (MOG) associated acute disseminated encephalomyelitis (ADEM) contributing to the HE in this patient [26].In another case study on HE patients, autoantibodies against the glutamate receptor ε2 subunit (GluRε2Ab) were found in CSF [20].(figure 2).Three studies focused on investigating the mechanism of brain abnormalities in HT.In the first study, authors investigated whether and how HT itself can trigger alterations in the formation of longterm potentiation (LTP) in the Schaffer collateral-CA1 pathway located in the hippocampus.The hippocampus is associated with cognitive function, so its alterations are associated with learning and memory abnormalities.LTP is recognized as one of the main mechanisms for storing memories in the brain.Studies on the hippocampus of euthyroid mice with HT showed decreased synaptic number, impaired synaptic structure, dysfunctional glutamate-glutamine cycle, and damage to LTP in the Schaffer collateral-CA1 pathway.The authors believe that the synaptic loss could be at least partially attributed to astrocyte damage [27] (figure 3).

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In the second study, an experimental euthyroid mouse model of HT was tested for anxietylike behavior, depression-like behavior, and neuroinflammation.Increased anxiety-and depressivelike behaviors were observed, accompanied by microglia activation, astrocyte activation, and increased expression of proinflammatory cytokines (interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)) in the frontal cortex.Additionally, proinflammatory cytokines may have activated certain key genes related to the serotonin system in HT euthyroid mice, resulting in reduced serotonin levels in J o u r n a l P r e -p r o o f the frontal cortex.This study demonstrates that even in the euthyroid state, HT continues to induce neuroinflammation and alters related serotonin signaling [28] (figure 2).
In the third study, five growth factors: brain-derived neurotrophic factor (BDNF), Stem Cell Factor (SCF), Insulin-like Growth Factor-Binding Protein 2 (IGFBP-2), Epidermal Growth Factor (EGF), and interleukin 7 (IL-7) were determined in serum of patients at risk for HT.64 TPOAb-negative females with at least one first-or second-degree relative were enrolled in the study.During the 5-year follow-up period, 32 of whom seroconverted to TPOAb positivity and 32 did not.EGF and IGFBP-2 levels were significantly higher, while BDNF levels was significantly lower in the study group compared to the control group.The SCF levels were significantly higher in the seroconversiv group compared to the seronegative group.After analyzing the relationship with other cytokines, these aberrancies were found to be similar to those found in patients with mood disorders.This suggests that shared growth and differentiation defects in both the hematopoietic and neuronal system may underlie thyroid autoimmunity and mood disorders [29] (figure 3).Bladowska and others found a potential biological association between thyroid dysfunction and cerebral metabolic changes.They assessed metabolic alterations within the normal-appearing brain in euthyroid patients with HT using magnetic resonance spectroscopy (MRS) and correlated MRS measurements with hormonal concentrations.No signs of central nervous system involvement were observed in patients.Authors suggest that MRS could serve as a sensitive biomarker capable of detecting early cerebral metabolic disturbances associated with HT [30].Subsequent research investigated the evoked potentials (EP) and magnetic resonance spectroscopy (MRS) in euthyroid patients with HT.One-third of the patients had changes in the brain bioelectrical activity without nervous system involvement.In 34% of the patients, abnormal VEP and BAEP results were recorded.No statistically significant correlations were found between the mean VEP parameters and the thyroid profile or the applied dose of L-thyroxine.However, a significant positive correlation was found J o u r n a l P r e -p r o o f between the TSH level and the wave BAEP III-V interpeak latency.Another study examined eventrelated potentials (ERP) and MRS, correlating the findings with cognitive function.The event-related potentials (ERP) revealed significantly prolonged N200 and P300 waveform latencies across all leads.The N-acetylaspartate/creatine (NAA/Cr) ratio correlated significantly negatively with all N200 latencies, while the myo-inositol/creatine (mI/Cr) ratio showed a significant positive correlation with P300 latencies.These findings indicate that prolonged response latencies are associated with metabolic changes in the cerebral cortex.The absence of significant differences in ERP and MRS parameters among patients with different initial diagnoses may suggest that the autoimmune process itself, rather than the severity of pre-therapy thyroid dysfunction, is responsible for the development of cognitive impairment.Authors suggest that both MRS and ERP could be a useful tool in assessing risk of developing cognitive impairment [31,32].Although EP and ERP are nonspecific, they may still contribute to the understanding of Hashimoto's thyroiditis.
In some Magnetic Resonance Imaging (MRI) studies, no cognitive or neural alterations were detected in euthyroid HT patients and the current mood status was not found to be related to depression-related networks.Moreover, in a thorough neuropsychological test battery encompassing memory, working memory, psychomotor speed, sustained attention, and attention lapses, along with an associative memory task conducted during fMRI acquisition, no evidence of cognitive deficits was observed.However, TPO autoimmunity and treatment duration showed a relationship between depression and hypothyroidism-related brain structure and function [33].Other studies indicate the existence of certain changes in MRI examinations of euthyroid patients with HT.Performance on the d2 test and MRI measured grey matter (GM) density of the left inferior frontal gyrus (LIFG) in euthyroid patients with HT, compared to a control group of euthyroid patients with other thyroid diseases, showed a significant correlation between GM density and d2 test of attention total score in HT group.Particularly low achievement in the d2 test was associated with reduced GM density of this brain region suggesting an influence of autoimmune processes on the frontal cortex in HT [34].
Diffusion tensor imaging (DTI) is becoming increasingly valuable for assessing microstructural brain involvement in a variety of neurological and psychiatric disorders.DTI revealed loss of axonal structural integrity and/or demyelination in white matter pathways in euthyroid patients with HT [35].
Technetium-99m ethyl cysteinate dimer single-photon emission computed tomography ( 99m Tc-ECD SPECT) of the brain showed irregular and patchy cerebral distribution in patients with HT.These abnormalities were similar to those observed in cases of severe HE [36].Electroencephalography (EEG) studies have not yet been conducted in patients with HT.However, it is worth noting that such studies have been performed in several case reports of patients with HE.These studies have demonstrated diffuse slowing of the background activity, repetitive focal spikes or sharps, interictal epileptiform discharges, photoparoxysmal response, photomyogenic response, and generalized biphasic or triphasic waves in EEG.Also myoclonus seizures were observed in HE patients.These findings were inconclusive; however, it would be valuable to investigate EEG in patients with HT to determine if any preliminary changes are present.[37].
The limited number and inconclusive nature of imaging studies conducted so far indicate that it is still a long way before they can be incorporated into the diagnostics of CNS disorders in HT.Nevertheless, several studies suggest that CNS changes are present, and it is worthwhile to expand this area of knowledge.Imaging studies could contribute to the understanding of the pathogenesis of CNS changes in patients with Hashimoto's thyroiditis.

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Impairments in cognitive functioning, mental and physical health, and general well-being were confirmed by numerous questionnaire studies [9].Due to this, these studies could serve as a straightforward method for screening and detecting the risk of developing CNS disorders in HT euthyroid patients.Some research suggests a relationship between these impairments and antithyroid antibodies.Behavioral problems correlated with TPOAb, while awareness level inversely correlated with TGAb [14].Additionally, TPOAb, TGAb, and TSH-RAb shared homology with proteins expressed in both CNS and thyroid: ENO1, AKR1A, and DDAHI [15,16].However, some studies found no correlation between TPOAb and cognitive impairment [17].It has been suggested that testing antithyroid antibodies in CSF rather than in serum could enhance diagnostic reliability in HT patients [18].Furthermore, antibody-specific indices (ASIs), which allow the differentiation between antibodies passively diffusing from the serum and brain-derived antibodies, could serve as potential biomarker for brain alterations in HT patients [19].It would be valuable to conduct similar studies on HT euthyroid patients to determine if there is a basis for suspecting the presence of these antibodies in their CSF.
Authors also indicate that other coexisting antibodies could be involved in brain alterations associated with HT and even in HT development.ENO1Ab can damage both thyrocyte and brain cells, causing the cognitive and behavioral impairments and/or autoimmune inflammatory responses against thyroid tissue [23].The anti-PDIA3 antibody is found involved in thyroidal and intracerebral damage through activating the complement system and presumably is responsible for HT development [24].MOGAb, GluRε2Ab, and CNSAb were found in HE or HT patients, but further studies in the euthyroid HT patient are necessary to investigate whether they contribute to CNS impairments [26].The discovery of new antibodies would not only enhance our understanding of the pathogenesis of CNS damage in HT but could also contribute to identifying new targets for targeted therapies.
Authors found alterations in the formation of long-term potentiation in the Schaffer collateral-CA1 pathway located in hippocampus, decreased synaptic number, impaired synaptic structure, and dysfunctional glutamate-glutamine cycle in euthyroid HT mice [27].Also, microglia activation, astrocyte activation, and increased expression of proinflammatory cytokines (IL-1β and TNF-α) resulting in more anxiety-and depressive-like behaviors were observed in HT euthyroid mouse models.Authors suggest that proinflammatory cytokines could activate some key serotonin-system-related genes, resulting in reduced frontal cortex serotonin levels [28].EGF, IGFBP-2, BDNF, and SCF in association with other cytokines could be responsible for both HT development and mood disorders [29].Available studies on the potential pathogenesis of CNS damage in HT address distinct aspects and have shown various changes, suggesting that the pathogenesis of CNS damage in HT might be multifactorial.
Finding imaging studies that reliably show CNS changes in euthyroid HT patients would enable objective detection of these changes.The authors suggest that MRS and ERP could be sensitive biomarkers useful in assessing risk of developing cognitive impairment associated with HT [30].Some MRI studies found no cognitive or neural alterations in euthyroid HT patients and current mood status wasn't related to depression-related networks [33].Another MRI study found a significant correlation between GM density and the d2 test of attention total score as well as reduced GM density of this brain region in patients with particularly low achievement in the d2 test, suggesting an influence of autoimmune processes on the frontal cortex in HT euthyroid patients [34].DTI in euthyroid patients with HT showed loss of axonal structural integrity and/or demyelination in white matter pathways [35].99mTc-ECD brain SPECT revealed that cerebral distribution was irregular and patchy in HT patients [36].Performing imaging tests in euthyroid patients with HT could be a reliable way to find patients at risk of developing CNS disorders.However, current studies are insufficient in number and need to be expanded before any procedural recommendations can be considered.

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In conclusion, research shows CNS disorders are present in HT patients despite effective compensation of thyroid hormone levels with levothyroxine.The etiology of CNS alterations in patients with HT in a euthyroid state remains poorly understood.However, current research indicating diverse changes suggests that it is a complex and multifactorial process.Questionaries could be an easy and effective way to find patients at risk of developing CNS disorders.Imaging studies would provide a more objective method.However, further research is necessary to create standards for MRI, MRS, or ERP diagnostics and for finding other imaging studies to diagnose brain disorders in HT patients.It is still unknown whether Hashimoto's encephalopathy (HE) is a consequence of Hashimoto's thyroiditis (HT).Nevertheless, it would be worthwhile to investigate the antibodies found in patients with HE in those with HT in a euthyroid state to determine if these antibodies might precede the onset of CNS alterations or encephalopathy.
Much research is still needed to understand the pathogenesis of CNS disorders in HT euthyroid patients and to find reliable biomarkers and treatment methods that could improve patients' quality of life.HT impairs the Schaffer collateral-CA1 pathway in the hippocampus of euthyroid mice.This impairment is associated with decreased synaptic number, damaged synaptic structure, and dysfunctional glutamate-glutamine cycle, leading to cognitive alterations.The authors attribute some of this damage to astrocyte dysfunction.27

Cai et al. (2018)
HT induces anxiety-like and depression-like behaviors in euthyroid mice, alongside neuroinflammation.This includes microglia and astrocyte activation and increased levels of proinflammatory cytokines (IL-1β and TNFα) in the frontal cortex.The study also notes reduced serotonin levels, suggesting that HT can alter serotonin signaling even in euthyroid states.28 J o u r n a l P r e -p r o o f

Table 1
The Key Findings from Studies Included in the Article Examined growth factors in women at risk for HT, showing significantly higher levels of EGF and IGFBP-2 and lower levels of BDNF in those who seroconverted to TPOAb positivity.Higher SCF levels were noted in those who seroconverted compared to seronegative controls.The findings suggest a link between growth factor aberrancies and mood disorders, potentially indicating shared defects in the hematopoietic and neuronal systems.
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