Tar DNA binding protein 43,a proteinopathy with preference for olfactory structures in COVID-19 subjects

Abstract

Background

Olfactory impairment (OI) is an early symptom of neurodegenerative diseases (ND) and COVID-19 infection. Proteinopathies associated with ND include amyloid-β (Aβ), hyperphosphorylated τ (HPτ), α-synuclein (α-syn), and Tar DNA binding protein 43 (TDP43). It is unclear whether COVID-19 infection influences the listed proteinopathies in the olfactory bulb and tract (OB/OT) aggravating the OI.

Objective

To study proteinopathies associated with ND in the brain and OB/OT in 32 subjects with COVID-19 infection and 10 age- and gender-matched controls.

Methods

Postmortem brain tissue was assessed for various proteinopathies and the OB/OT for proteinopathies, inflammatory markers and a marker for severe acute respiratory syndrome coronavirus 2 spike protein.

Results

Twenty percent of control and 16% of COVID-19 subjects lacked proteinopathies in their OB/OT. HPτ was detected in OB/OT in 80% of controls and 81% of COVID-19 subjects, Aβ in 30% of controls and 16% of COVID-19 subjects. All controls lacked TDP43 in OB/OT, 40% displayed TDP43 in their brain. TDP43 was seen in the OB/OT in 38% of COVID-19 subjects, of whom 42% lacked TDP43 in the brain. Sixty percent of controls displayed α-syn in OB/OT and the brain, whereas 34% of COVID-19 subjects displayed α-syn in the OB/OT, of whom 36% lacked it in the brain.

Conclusions

All proteinopathies associated with ND were detected in OB/OT in COVID-19 patients whereas TDP43 was lacking in controls. Our results suggest that there might be an association between COVID-19 and TDP43 and α-syn in the OB/OT, which may explain the chronic OI.

Keywords

α-synuclein Alzheimer's disease amyloid-β COVID-19 hyperphosphorylated-τ neurodegeneration olfactory bulb olfactory impairment Tar DNA binding protein 43

Introduction

The Coronavirus disease 2019 (COVID-19) pandemic changed our world. With almost 800 million infections and over 7 million deaths, it has had a profound impact not only on individuals and their relatives, but also on healthcare systems nationally and globally.¹ COVID-19 is primarily associated with acute respiratory symptoms, but many patients also suffer from vascular, cardiac, and neuropsychiatric manifestations in parallel with respiratory failure.² During the acute phases of the disease, the majority of COVID-19 patients develop neurological symptoms, i.e., olfactory impairment (OI), encephalopathy, cerebrovascular disease, and encephalitis.^2,3 Additionally, many individuals develop long-term neurological symptoms, such as brain fog, headaches, sleep abnormalities, and persistent OI, which can last for years.^2,3

OI, i.e., anosmia, hyposmia, and parosmia, is described as an early and, by some authors, a cardinal symptom of COVID-19 that, in the majority of cases, disappears after 2–3 weeks.^2,4,5 Unfortunately, a substantial number of individuals develop longstanding OI that persist for over 12 months.^2,5 The pathology behind the OI is not entirely understood.

The COVID-19 infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), which does not seem to be neurotropic. There are several neuropathological studies assessing SARS-COV-2 in the brain and olfactory structures.^5–15 Nevertheless, there are very few studies where SARS-COV-2 has been detected within the neurons or glial cells.^7,8,10 The olfactory bulb (OB) and olfactory tract (OT) seem to be affected by presumed secondary changes related to COVID-19 infection. The main pathological findings in the olfactory structures include astro- and microgliosis, whereas the SARS-COV-2 itself is predominantly detected in the respiratory and olfactory mucosa.^6,15–17

The assessment of brain pathology in individuals with COVID-19 has revealed edema, hypoxic/ischemic injuries, microvascular/vascular damage, leading to petechial bleeding and hemorrhages. Additionally, astroglial- and microglial activation, mostly affecting the brainstem, has been observed, along with lymphocytic infiltrates in the perivascular space and, albeit sparsely, the intraparenchymatous.^{7,9,11–14,17}

It has been reported that a substantial number of individuals with COVID-19 have also displayed signs of age-related neurodegeneration, i.e., Alzheimer's disease neuropathological change (ADNC), Lewy body disease (LBD), and Limbic-predominant Age-related TDP43 Encephalopathy NC (LATE-NC).^11–13,17 Noteworthy, neuroinflammation has been reported in all of these neurodegenerative diseases, even before the COVID-19 pandemic. Thus, both neurodegeneration and COVID-19 infection are associated with neuroinflammation.^17–23

Additionally, it has been reported that the altered proteins associated with the most common neurodegenerative diseases, i.e., amyloid-β (Aβ), hyperphosphorylated τ (HPτ), α-synuclein (α-syn), and Tar DNA-binding protein 43 (TDP43), can be detected in the OB and OT.^24–27 α-syn has been detected in the OB and OT early in the disease process of LBD, and OI is an early symptom of the disease.^25,27 In contrast, ADNC and TDP43 have been detected in olfactory structures later in the disease and are associated with dementia-related illness, which includes neurological symptoms such as OI.^24–28 This is particularly intriguing, as mechanisms associated with both neurodegeneration and COVID-19 infection may be synergistic. Moreover, COVID-19 infection may accelerate neurodegenerative processes, contributing to further disease progression.^{6,14,15,17,21–23,29}

The objective of this study was to assess the olfactory pathology in association with COVID-19 infection and neurodegeneration.

Methods

This study was approved by the local ethics committee (Dnr 2011/286 and updated 2015).

Between 2020 and 2021, 322 PM brains underwent neuropathological examination at Uppsala University Hospital (UUH). Among these, 285 cases were autopsied locally at UUH, 18 at local hospitals, and 19 at the department of forensic medicine. COVID-19 infection was verified with polymerase chain reaction (PCR) postmortem, prior to autopsy in 39 cases. In subjects hospitalized prior to death, a PCR analysis was performed when the patient was admitted to the hospital as well. The duration of the infection ranged from acute to subacute. Some subjects were referred to autopsy from home, by a general practitioner, with unknown cause of death and others were referred to autopsy from a hospital ward, as they were cared for COVID-19 or other diseases. Seven cases lacked OB or OT samples for neuropathological assessment and were excluded. Thus, 32 cases were included in this study.

Ten age- and gender-matched control cases with available OB and OT samples for neuropathologic examination were chosen as controls. These subjects represented general population and thus displayed various age-related protein alterations within their brain tissue.

The clinical parameters prior to death of all subjects included in this study are listed in Supplemental Table 1.

Regardless of the autopsy site, the brains were removed at the autopsy, weighed, and placed in 10% buffered formalin for fixation. All neuropathological assessments were performed at UUH. The OB and OT were removed from the base of the brain prior to the sectioning of the brain. The brain was then cut into 1 cm-thick coronal slices, assessed for macroscopic alterations, and sampled as previously described.³⁰ All samples, including the OB and OT, were processed into paraffin blocks and cut into 7 µm-thick sections for hematoxylin-eosin and immunohistochemical (IHC) staining.

All stainings were performed using an automated platform according to manufacturer's instructions. All Abs used were commercially sourced and are listed in Table 1.

Table 1.

Immunohistochemical stains.

Antibody	Clone	Company/Code	Dilution	Pretreatment
α-Synuclein	KM51	Novocastra/NCL-ASYN	1:100	pH High + 98–100% FA
Amyloid β	4G8	Biolegend/800703	1:1000	98–100% FA-2 min
CD3	polyclonal	Dako/GA503		pH Low
CD20	L26	Dako/GA604		pH High
Human leucocytic antigen -DR, α-chain (HLA-DR)	TAL.1B5	Dako/M0746	1:30	pH Low
Ionized calcium-binding adaptor molecule1(Iba1)	polyclonal	Wako/NordicBiolabs 019-19741	1:5000	pH High
Phosphorylated (pS409/410) transactive DNA binding protein 43 (pTDP43)	11–9	CosmoBio/TIP-PTD-M01	1:5000	ac, CB
SARS-CoV-2 (COVID-19) spike protein	1A9	GeneTex/GTX632604	1:200	pH High
Hyperphosphorylated (Ser202/Thr205) τ (TAU8)	PHF-TAU-AT8	Fisher Sientific-Invitrogen/MN1020	1:1000

For α-synuclein, amyloid-β, Tau 8, HLA-DR and Iba1 Dako Autostainer Plus (Dako Cytomation) and for CD3, CD20, CD68, GFAP, MAP2 and synaptophysin Dako OMNIS were used. Autoclave (ac), formic acid (FA), Tris-EDTA buffer pH 9.0 (TE), citrate buffer pH6.0 (CB).

The protein alterations in the brain associated with neurodegenerative diseases, i.e., HPτ, Aβ, TDP43, and α-syn, were assessed and staged according to international consensus criteria.^31–35 The extent of altered proteins in the brain of all subjects included in this study are listed in Supplemental Table 2.

The altered proteins were assessed in the OB and OT as present or not, i.e., dichotomized. In addition to the neurodegenerative markers, inflammatory markers, i.e., microglial (HLA-DR and Iba1), lymphocyte markers (CD3 and CD20), and an antibody targeting the COVID-19 spike protein, were assessed for presence or absence.

Statistical analyses were performed using IBM SPSS statistics software, version 28. Non-parametric tests were applied as the continuous variables are not normally distributed. The mean ± standard error of the mean (m ± SE) was calculated to describe the cohort. Statistical differences between the study groups were assessed using the Mann Whitney-U (MWU). Fisher's exact test (FE) and the Pearson Chi-Squire (PCS) test were used to analyze the contingency of categorical data. Spearman's correlation test was used to examine correlations between the studied variables.

Results

In total, 42 cases were included in this study: 32 with COVID-19 infection and 10 age- and gender- matched controls. The demographics of the study population are listed in Table 2. The mean age of the study population was 70 ± 2 years. The age at death of the COVID-19 cases did not differ significantly from the controls. Clinical dementia diagnosis was present in 20% of the control group, compared to 13% in the COVID-19 group.

Table 2.

Demographics.

	All	Control	COVID-19	Statistics MWU/FE
All	42	10	32	-/-
Male / female	28 / 14	5 / 5	23 / 9	-/ns
Age at death m ± SE	70 ± 2	70 ± 5	70 ± 2	ns/-
With dementia	6	2	4	-/ns
With dementia (male/female)	4/2	1/1	3/1	ns/ns

m ± SE, mean ± Standard Error of means; CVD: cardiovascular disease; MWU: Mann Whitney U test; FE: Fisher's Exact test.

Brain pathology

The incidence and extent of HPτ, Aβ, TDP43, and α-syn in the brains of all 42 subjects and each study group, are summarized in Table 3. There were no significant differences between the groups regarding the incidence or extent of HPτ, Aβ, and TDP43. The incidence and extent of α-syn were higher in the control group (6 out of 10 controls) compared to 7 out of 32 subjects with COVID-19. Noteworthy, 2 (20%) subjects in the control group and 2 (6%) of COVID-19 subjects were demented and displayed high level of α-syn pathology.

Table 3.

Incidence and extent of altered proteins in the brain.

	All n (%)	Controls n (%)	COVID19 n (%)	Statistics FE/PCS/MWU
	42	10	32
HPτ	41 (98)	10 (100)	31 (97)	-/ns/ns
HPτ only seen in LC	8 (19)	3 (30)	5 (16)
HPτ Braak stage I-II	21 (50)	2 (20)	19 (59)
HPτ Braak stage III-IV	9 (21)	4 (40)	5 (16)
HPτ Braak stage V	3 (7)	1(1)	2 (6)
Glial HPτ, ARTAG	16 (38)	5 (50)	11(34)	ns
Aβ	32 (76)	6 (60)	26 (81)	ns/ns/ns
Aβ Thal phase 1–2	17 (41)	3 (30)	14 (44)
Aβ Thal phase 3–4	10 (24)	1 (10)	9 (28)
Aβ Thal phase 5	5 (12)	2 (20)	3 (9)
CAA	20 (48)	3 (30)	17 (53)	ns
TDP43	20 (48)	4 (40)	16 (50)	ns/ns/ns
TDP43 Josephs stage 1	3 (7)	0	3 (9)
TDP43 Josephs stage 2	15 (36)	4 (40)	11 (34)
TDP43 Josephs stage 3	2 (5)	0	2 (6)
α-syn	13 (31)	6 (60)	7 (22)	0.032/0.029/ns
α-syn Braak stage 2	2 (5)	1 (10)	1 (3)
α-syn Braak stage 3	2 (5)	2 (20)	0
α-syn Braak stage 4	2 (5)	0	2 (6)
α-syn Braak stage 6	7 (5)	3 (30)	4 (13)

FE: Fisher's Exact test; PCS: Pearson Chi-Square; MWU: Mann Whitney U test; HPτ: hyperphosphorylated τ; ARTAG: age related tau astrogliopathy; Aβ: amyloid-β; CAA: cerebral amyloid angiopathy; TDP43: transactive DNA binding protein 43; α-syn: α-synuclein.

Neurofibrillary/HPτ pathology was detected in 41 out of 42 cases. The majority of subjects displayed low levels of HPτ pathology, with 29 (69%) cases (5/50% controls and 24/75% COVID-19 cases) displaying HPτ only in the locus coeruleus or at Braak stage I-II. High levels of HPτ pathology, i.e., Braak stage V, were observed in 3 (7%) of the subjects (1/10% controls and 2/6% of COVID-19 cases). Age-Related Tau AstroGliopathy (ARTAG) was present in 16 (38%) cases (5/50% controls and 11/34% COVID-19). Aβ was detected in the brain parenchyma of 32 (76%) cases (6/60% controls and 26/81% COVID-19), and cerebral amyloid angiopathy was found in 20 (48%) cases (3/30% controls and 17/53% COVID-19). TDP43 pathology was seen in 20 (48%) cases (4/40% controls and 16/50% COVID-19), with the majority 15 (36%) cases (4/40% controls and 11/34% COVID-19) classified as Josephs stage 2. α-syn was identified in 13 (31%) subjects, with a higher prevalence in controls 6 (60%) compared to the COVID-19 group 7 (22%).

Only 3 (7%) subjects (2/20% controls and 1/3% COVID-19) displayed HPτ pathology only, whereas 38 subjects displayed concomitant proteinopathies within their brains. Concomitant pathologies in the whole cohort, as well as in the control and COVID-19 group, are visualized in a Venn diagram (Figure 1).

Figure 1.

Venn diagram visualizing concomitant pathologies in the control and COVID-19 groups. The upper row represents pathologies in the brain, while the lower row represents the pathologies in the olfactory structures. Tau: hyperphosphorylated τ; Beta: amyloid-β; TDP: transactive DNA binding protein 43; Syn: α-synuclein.

The pathology of the olfactory bulb and tract

Inflammatory markers

Reactive changes were seen within the OB and OT in hematoxylin-eosin stained sections, including gliosis and numerous corpora amylacea (CA) in all 42 subjects. CA were predominantly found in the subpial neuropil within both the OB and OT. Additionally, numerous, microglial cells were observed applying Iba1 and HLA-DR antibodies in all cases, with only focal microglial nodules present. CD3 reactive T-lymphocytes were sparse, seen mostly in close proximity to blood vessels. However, CD20 lymphocytes were absent.

When applying the SARS-spike antibody, labeling was seen in association with the CA. Additionally, perinuclear, dot-like labeling was seen in glial cells. The labeling was observed in all subjects and was interpreted as unspecific, thus not taken into consideration in the final analyses. The reactive changes within the OB/OT, as well as the outcomes of inflammatory markers and the SARS-spike antibody, are visualized in Figure 2.

Figure 2.

Photomicrographs of stained sections from the olfactory bulb and tract (OB/OT) of a subject with COVID-19 infection. (a-c) Routine hemotoxylin-eosin stained sections; (d-g) immunohistochemically stained sections. (a) the OB/OT; (b) the anterior olfactory nuclei; (c) numerous corpora amylacea in the OB/OT. Microgliosis visualized using an antibody (ab) against human leucocytic antigen -DR in (d) and ionized calcium-binding adaptor molecule1 in (e). A few T-lymphocytes associated with a vessel, visualized by CD3 ab in (f). (g) Immunohistochemical staining using an ab against the SARS-CoV-2 (COVID-19) spike protein. Note the labeling around corpora amylacea (black arrow) and the dot-like perinuclear staining in proximity to the cell nuclei (white arrows). Bar in (a) 5 mm, in (b) 200 µm, in (c) 50 µm, in (d) and (e) 100 µm, in (f) 50 µm and (g) 20 µm.

The incidence of neurodegeneration associated proteinopathies

The incidence and extent of HPτ, Aβ, TDP43, and α-syn within the OB and OT of all 42 subjects is summarized in Table 4.

Table 4.

Incidence of altered proteins in the olfactory bulb and nerve.

	All n (%)	Controls n (%)	COVID19 n (%)	Statistics FE
	42	10	32
HPτ	34 (81)	8 (80)	26 (81)	ns
Glial HPτ	21(50)	7 (70)	14 (44)	ns
Aβ	8 (19)	3 (30)	5 (16)	ns
TDP43	12 (29)	0	12 (38)	0,02
α-syn	17 (41)	6 (60)	11 (34)	ns

FE: Fisher's Exact test; HPτ: hyperphosphorylated τ; Aβ: amyloid-β; CAA: TDP43: transactive DNA binding protein 43; α-syn: α-synuclein.

Neurofibrillary/HPτ pathology was present in 34 (81%) subjects (8/80% controls and 26/81% COVID-19), whereas glial HPτ was seen in 21 (50%) cases (7/70% controls and 14/44% COVID-19). The neurofibrillary HPτ pathology appeared as tangles in gray matter areas within the OB, as well as in processes within the gray and white matter of the OB and OT. Glial HPτ was seen solely in astrocytes. Aβ was found in 8 (19%) cases (3/30% controls and 5/16% COVID-19), restricted to gray matter areas of the OB. α-syn pathology was seen in 17 (41%) cases (6/60% controls and 11/34% COVID-19), appearing both as intracellular and neuritic inclusions. There was no significant difference in the incidence of HPτ, Aβ, or α-syn between the control and COVID-19 groups (Table 4).

There was a significant difference in TDP43 pathology, which lacked in the control group but was detected in 12 (38%) of the COVID-19 group (FE = 0.02). The different proteinopathies within the OB and OT are visualized in Figure 3.

Figure 3.

Photomicrographs of immuohistochemically stained sections from the olfactory bulb and tract (OB/OT) from subjects with COVID-19. In (a-b), applying antibody (ab) against hyperphosphorylated τ; note in (a), neurofibrillary pathology within the anterior olfactory nuclei and the surrounding tract and in (b), astroglial HPτ pathology. In (c), applying ab against amyloid-β and in (d) ab directed against α-synuclein. In (e-f), neuronal perinuclear staining applying an antibody against transactive DNA binding protein 43. Bar in (a-d) 100 µm and (e-f) 50 µm.

Seven (17%) subjects, (2/20% controls and 5/16% COVID-19), lacked protein alterations in their OB/OT. Thirteen (31%) cases (2/20% controls and 11/34% COVID-19) displayed one protein alteration, i.e., 12 (29%) cases (2/20% controls and 10/31% COVID-19) with HPτ- pathology and 1 case with TDP43- pathology in the COVID-19 group (3%). Concomitant pathologies were observed in the remaining 22 (52%) cases (6/60% controls and 16/50% COVID-19). The distribution of proteinopathies within the OB and OT in the control and COVID-19 groups is illustrated in Figure 1.

The association between brain proteinopathies and the proteinopathies within the olfactory bulb and tract

The correlations between the brain proteinopathies and those within the OB/OT in the whole cohort, as well as in the control and COVID-19 group, are summarized in Table 5.

Table 5.

Spearman’ rho (r) correlations between the assessed pathologies within the brain and olfactory bulb. Correlation shown when significance <0.05¹, < 0.01².

			All	Controls	COVID-19
	number of cases		42	10	32
Between altered proteins within the brain	Age	HPτ	0.61²		0.57²
	Age	Aβ	0.41²	0.64¹
	Age	TDP43	0.69²	0.85²	0.62²
	Age	α-syn	0.39¹	0.71¹
	HPτ	Aβ	0.60²	0.74¹	0.53²
	HPτ	TDP43	0.76²		0.80²
	HPτ	α-syn	0.58²	0.65¹	0.52²
	Aβ	TDP43	0.40²		0.42¹
	Aβ	α-syn	0.48²		0.51²
	TDP43	α-syn	0.46²	0.75¹	0.43¹
Between altered proteins within the olfactory bulb	Age	HPτ	0.61²		0.66²
	Age	Aβ	0.36¹
	Age	TDP43			0.36¹
	Age	α-syn	0.44²		0.46²
	HPτ	Aβ	0.60²	0.72¹	0.53²
	HPτ	TDP43			0.41¹
	HPτ	α–syn	0.54²		0.53²
	Aβ	TDP43
	Aβ	α-syn	0.41²
	TDP43	α-syn			0.43¹
Between altered proteins within the brain (B) and within the olfactory bulb (O)	HPτ in B	HPτ in O	0.74²	0.73¹	0.75²
	Aβ in B	Aβ in O	0.65²	0.83²	0.56²
	TDP43 in B	TDP43 in O
	α-syn in B	α-syn in O	0.82²	0.95²	0.70²
	HPτ in B	Aβ in O	0.50²	0.74¹	0.38¹
	HPτ in B	TDP43 in O
	HPτ in B	α-syn in O	0.62²		0.59²
	Aβ in B	HPτ in O	0.63²	0.76¹	0.59²
	Aβ in B	TDP43 in O
	Aβ in B	α-syn in O
	TDP43 in B	HPτ in O	0.55²		0.65²
	TDP43 in B	Aβ in O	0.38¹		0.41¹
	TDP43 in B	α-syn in O	0.46²		0.47²
	α-syn in B	HPτ in O	0.57²		0.58²
	α-syn in B	Aβ in O	0.48²	0.65¹	0.39¹
	α-syn in B	TDP43 in O

HPτ: hyperphosphorylated τ; Aβ: amyloid-β; TDP43: transactive DNA binding protein 43; α-syn: α-synuclein.

The HPτ pathology was present in the brain of 41 subjects (all controls and 31/97% of COVID-19 cases), whereas in the OB/OT, neuronal HPτ was seen in only 34 (81%) cases (8/80% of controls and 26/81% of COVID-19 cases). The glial HPτ – ARTAG was seen in 16 subjects (38%) in the brain (5/50% of controls and 11/34% of COVID-19 cases), whereas astroglial HPτ was detected in the OB/OT of 21subjects (50%) (7/70% of controls and 14/44% of COVID-19 cases). Aβ was seen in the brain of 32 subjects (76%) (6/60% of controls and 26/81% of COVID-19) and in the OB/OT in 8 subjects (19%) (3/30% controls and 5/16% COVID-19). TDP43 was seen in the brains of 20 subjects (48%) (4/40% controls and 16/50% of the COVID-19 cases) and in the OB/OT of 12 subjects, but only in the COVID-19 group (38%). Five (16%) COVID-19 positive subjects displayed TDP43 pathology within their OB/OT but lacked TDP43 pathology in their brains. α-syn pathology was seen in the brains of 13 subjects (31%) (6/60% of the controls and 7/22% of COVID-19), whereas it was found in the OB/OT in 17 cases (41%) (6/60% controls and 11/34% COVID-19 subjects). In the COVID-19 group, 4 subjects displayed α-syn pathology within the OB/OT, but not in the brain.

Discussion

Olfactory impairment is one of the most common symptoms of a COVID-19 infection, which can often result in longstanding sensory olfactory symptoms, such as anosmia and hyposmia.^2,5 Aged individuals, especially those with ongoing neurodegenerative diseases, are more vulnerable to COVID-19 infection.^11–13,17 Additionally, some studies suggest that persistent OI after COVID-19 infection is more common in subjects with neurodegenerative diseases.^6,21,22

Here, we assessed the most common proteinopathies associated with neurodegenerative diseases, i.e., Aβ, HPτ, α-syn, and TDP43, in post-mortem brains and olfactory structures from 42 subjects: 32 deceased individuals with ongoing COVID-19 infection and 10 age- and gender-matched controls.

The total incidence of Aβ, HPτ, α-syn, and TDP43 pathologies in the postmortem brains of subjects in the current study aligns with findings previously described by us in a large cohort of postmortem brains from 1610 subjects.³⁶ This supports the idea that, despite our study's limited number of subjects, it can be considered representative. Noteworthy, in our center the autopsy frequency is 12% and we assess brains of some 30% of all who come to autopsy, thus the brains of some 5% of all deceased have been assessed.

There was no significant difference in the incidence or extent of Aβ, HPτ, and TDP43 in the brain between the control group and COVID-19 group. Noteworthy, there was a difference between these two groups when assessing the incidence and extent of α-syn, which was higher in the control group. This outcome is probably related to a selection bias.³⁷

Consistent with previous reports, the most common proteinopathies associated with neurodegenerative diseases, i.e., Aβ, HPτ, α-syn, and TDP43, were detected in varying extents in the OB/OT in a substantial number of subjects, with 83% in our cohort.^15,24–27

There was no significant difference between the COVID-19 and the control groups regarding the HPτ and Aβ pathology within the OB/OT. ADNC correlated with age and all proteinopathies within the brain.

Olfactory impairment is associated with AD and is described as worsening with the severity of the disease.^38–40 HPτ, in particular, is associated with changes in smell perception in aging and in AD.^38,39 The extent of HPτ pathology in the OB/OT is associated with the severity of HPτ within the brain.^40,41 In addition, the presence of Aβ in the olfactory structures is independently associated with a higher risk of dementia.²⁴ However, we could not see any difference in the incidence of HPτ or Aβ in the OB/OT of subjects with or without ongoing COVID-19 infection. This would indicate that eventual olfactory symptoms, previously described in subjects with COVID-19, would probably not be associated with ADNC.^2,4,5

Interestingly, 7 (70%) control subjects and 14 (44%) subjects in the COVID-19 group displayed astroglial HPτ pathology, within their OB/OT. In contrast, only 5 (50%) control- and 11 (34%) COVID-19 subjects showed ARTAG in the brain. In line with our observation, a recent study reported glial HPτ pathology in the olfactory structures.⁴²

α-syn was detected in the OB/OT in 60% of the controls and 34% of the COVID-19 cases. The α-syn pathology within the OB/OT correlated with age, Aβ, and HPτ within the OB/OT, as well as in the brain with HPτ, α-syn, and TDP43. In the COVID-19 group, α-syn was seen in the OB/OT in five cases that did not display α-syn in their brain, whereas all control subjects displaying α-syn in their brain also exhibited the pathology in their olfactory structures. OI is a prodromal symptom in 90% of LBD cases, and hyposmia is associated with a 10% increased risk of developing PD.³⁸ α-syn is detected in the olfactory structures in LBD, which are one of the primary sites in the central nervous system, mirroring OI as one of the earliest symptoms of the disease.^27,38,43 Noteworthy, some studies have associated viral infections as triggers for α-syn pathology.^21,27,44 In our setting, the duration of COVID-19 infection was, to our knowledge brief, whereas it probably takes years to develop α-synucleinopathy. The presence of α-syn in the olfactory structures of subjects with COVID-19, but without LBD in the brain, suggests that subjects with α-syn in their OB/OT might be more susceptible to COVID-19 infection. This might explain the significant association between COVID-19 infection and an increased risk of new-onset PD in post COVID-19 subjects.^21,45 An upregulation and increased accumulation of α-syn have been described in association with COVID-19 infection in animal studies, as has been previously observed for other virus infections, which could promote the pathology further into a symptomatic disease.^46–48

TDP43 was detected within the brains of 40% of the controls and 50% of the COVID-19 subjects. Interestingly, only subjects in the COVID-19 group displayed TDP43 pathology within the OB/OT (38%), and five of these (42%) did not show TDP43 pathology in their brains. The subjects with TDP43 pathology only in their OB/OT were younger (although not significantly, MWU p = 0.073) compared to those who displayed TDP43 pathology in both their brain and OB/OT. TDP43 is associated with several neurodegenerative diseases, such as Frontotemporal lobar degeneration (FTLD), Amyotrophic lateral sclerosis (ALS), and AD.^49–51 The significance of the TDP43 proteinopathy in association with AD and hippocampal sclerosis has grown over the last decade, resulting in a new entity, i.e., Limbic predominant age-related TDP43 encephalopathy (LATE), established in 2019.^51,52 Similar to other neurodegeneration-associated proteinopathies, TDP43 affects different brain regions in a predilected manner and particularly amygdala, but olfactory localization has not been previously considered in staging.^35,51,53 Since OI is associated with neurodegenerative diseases, the prevalence of TDP43 in the OB/OT is of particular relevance. To our knowledge, there are only three previous studies assessing TDP43 within the olfactory structures in association with AD and ALS.^26,54,55 In these studies, TDP43 pathology was seen in the hippocampus in all cases, with higher density than in the OB/OT, suggesting that TDP43 accumulates in the olfactory structures later in the disease process.^26,54 In contrast to previous results, in our setting, 42% of COVID-19 subjects with TDP43 pathology in the OB/OT did not display TDP43 pathology in their limbic structures, i.e., the primary site of TDP43 proteinopathy in these cases. This could indicate that TDP43 pathology within the olfactory structures could be caused by different pathological mechanisms, as previously discussed, due to the differences in the frequency of TDP43 accumulation between AD and ALS cases.^26,54 Interestingly, the SARS-COV-2 virus encodes proteins involved in TDP43 cleavage, promoting increased aggregation and cytotoxicity.⁵⁶ The SARS-COV-2 spike protein binds directly to TDP43 RNA recognition motif proteins, thus directly altering the protein synthesis.⁵⁷ Additionally, TDP43 levels were significantly elevated in the serum of COVID-19 subjects and were associated with ferritin levels, a marker of inflammation, suggesting that increased levels of this protein may be triggered by SARS-COV-2.⁵⁸ Whether these mechanisms influenced the outcome of our results, i.e., TDP43 solely in the olfactory structures in our COVID-19 group, is unclear. However, our findings suggest that subjects with TDP43 pathology in their OB/OT may be more susceptible to viral (SARS-COV-2) infections or that the COVID-19 infection might influence TDP43 pathology. This pathology is progressive and may contribute to the chronic symptoms of OI.

Thus, in our setting, all proteinopathies associated with neurodegenerative diseases were detected within the OB/OT. TDP43, in particular, displayed an unusual distribution within the structures of the CNS in subjects with COVID-19. Neuroinflammation is an acknowledged mechanism involved in neurodegenerative diseases, and viral infections have been described to trigger neurodegeneration.^44,59 There are several studies demonstrating that the SARS-COV-2 virus triggers the formation and aggregation of altered proteins within the CNS, promoting neurodegenerative processes and diseases.^45,57,60,61 The OB/OT is connected to limbic structures through defined olfactory pathways, and the involvement of the olfactory nerve in different neurodegenerative diseases is well recognized.^{26,40,41,43,54,62} Based on our results, the incidence of TDP43 pathology in OB/OT differs in subjects with and without COVID-19. This outcome might be due to the viral involvement in the respiratory and olfactory mucosae. However, this hypothesis should certainly be investigated further.¹⁶

The reactive inflammatory response observed in the OB/OT was characterized by microgliosis, gliosis, and numerous corpora amylacea, but only a few CD3 reactive T-lymphocytes were seen. Numerous CA and sparse CD3 responses have previously been described in olfactory structures in subjects with COVID-19 infection, which is in line with our findings.^13,15,17 The microglial activation in our cases was diffuse and strong in all subjects, consistent with previous studies. However, other authors have reported a more patchy and subtle microglial reaction in subjects with COVID-19 infection.^7,13,15,17 The contradictory results may be related to various stages of COVID-19 infection that are seldom assessable. Furthermore, generalized microgliosis in the olfactory structures could be explained by the proximity to the nasal cavity, which serves as an entry route for external pathogens.^6,16 Another contributing factor may be the choice of antibody used to visualize the microglia. In most other studies, microgliosis within the OB/OT was assessed using CD68 (clone PG-M1), a lysosomal marker involved in phagocytic activity, which labels macrophages more readily and microglia to a lesser extent. In contrast, we assessed microglia using two markers, i.e., Iba1 and HLA-DR, both of which are associated with microglial activity in the CNS.^15,17,63,64

Since OI is one of the earliest symptoms of COVID-19, we assessed the prevalence of SARS-COV-2 spike protein within the OB/OT.^2,4,5 To our surprise, labeling was observed when applying the antibody against the SARS-COV-2 spike protein in both the control and COVID-19 groups. The labeling was associated with CA and appeared as dot-like perinuclear staining in glial cells (Figure 2). However, since the labeling was seen in both study groups, it was interpreted as unspecific. Thus, consistent with several publications, we could not confirm the presence of the SARS-COV-2 virus in the OB/OT or within the olfactory structures.^6,15,16

One of the weaknesses in our study is a limited number of control subjects. During the pandemics the autopsy frequency declined and the olfactory structures are not sampled by us routinely.

In summary, we assessed brain tissue and olfactory structures from 32 subjects who died with an ongoing COVID-19 infection and 10 age- and gender-matched controls. Our findings showed that all proteinopathies associated with neurodegenerative diseases, i.e., Aβ, HPτ, α-syn, and TDP43, were detected in the olfactory structures, and there were no significant differences in the incidence or extent of ADNC or LBD NC between these two groups. Interestingly, we also observed TDP 43 exclusively in the OB/OT of 12 subjects with COVID-19, a finding not previously reported. This suggests that individuals with TDP43 in their olfactory structures may be more prone to developing COVID-19 associated symptoms. Additionally, 42% of individuals in this group displayed TDP43 solely in the OB/OT, indicating that, in these subjects, this was the primary site of the pathology, not previously suggested. Moreover, 13% of subjects in this group displayed α-syn in their OB/OT, while they lacked this pathology in their brains, suggesting that this might be an early stage of LBD.

The inflammatory response within the OB/OT was dominated by gliosis and microgliosis, reflecting the primary line of defense against external pathogens in the olfactory structures. Consistent with most previous studies, we did not detect the SARS-COV-2 virus in the OB/OT of subjects in our cohort.

Supplemental Material

sj-docx-1-alr-10.1177_25424823251386016 - Supplemental material for Tar DNA binding protein 43, a proteinopathy with preference for olfactory structures in COVID-19 subjects

Supplemental material, sj-docx-1-alr-10.1177_25424823251386016 for Tar DNA binding protein 43, a proteinopathy with preference for olfactory structures in COVID-19 subjects by Sylwia Libard and Irina Alafuzoff in Journal of Alzheimer's Disease Reports

Footnotes

Acknowledgements

The subjects included in this study and/or their relatives provided consent for autopsy. We thank Meena Strömqvist for her critical reading of the manuscript.

ORCID iDs

Sylwia Libard

Irina Alafuzoff

Ethical considerations

This study was approved by the local ethical committee (Dnr 2011/286 and updated 2015).

Consent to participate

Not applicable

Consent for publication

Not applicable

Author contribution(s)

Sylwia Libard: Conceptualization; Formal analysis; Formal analysis; Investigation; Methodology; Project administration; Writing – original draft; Writing – review & editing.

Irina Alafuzoff: Conceptualization; Formal analysis; Investigation; Methodology; Writing – original draft; Writing – review & editing.

Funding

This study was funded by local grants from Uppsala University Hospital and the Märta Lundqvist Foundation.

Märta Lundqvists Stiftelse, Akademiska Sjukhuset.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The data is available and shared.

Supplemental material

Supplemental material for this article is available online.

References

World Health Organization . WHO COVID-19 dashboard, https://data.who.int/dashboards/covid19/cases?n=c (2024).

Mehandru

Merad

. Pathological sequelae of long-haul COVID. Nat Immunol 2022; 23: 194–202.

Bhola

Trisal

Thakur

, et al. Neurological toll of COVID-19. Neurol Sci 2022; 43: 2171–2186.

Iravani

Arshamian

Lundstrom

. Loss of olfactory sensitivity is an early and reliable marker for COVID-19. Chem Senses 2022; 47: bjac022.

Liang

Wang

. COVID-19 anosmia: high prevalence, plural neuropathogenic mechanisms, and scarce neurotropism of SARS-CoV-2? Viruses 2021; 13: 2225.

Xydakis

Albers

Holbrook

, et al. Post-viral effects of COVID-19 in the olfactory system and their implications. Lancet Neurol 2021; 20: 753–761.

Matschke

Lutgehetmann

Hagel

, et al. Neuropathology of patients with COVID-19 in Germany: a post-mortem case series. Lancet Neurol 2020; 19: 919–929.

Stein

Ramelli

Grazioli

, et al. SARS-CoV-2 infection and persistence in the human body and brain at autopsy. Nature 2022; 612: 758–763.

DeMarino

Lee

Cowen

, et al. Detection of SARS-CoV-2 nucleocapsid and microvascular disease in the brain: a case report. Neurology 2023; 100: 624–628.

10.

Emmi

Rizzo

Barzon

, et al. Detection of SARS-CoV-2 viral proteins and genomic sequences in human brainstem nuclei. NPJ Parkinsons Dis 2023; 9: 25.

11.

Eschbacher

Larsen

Moyer

, et al. Neuropathological findings in COVID-19: an autopsy cohort. J Neuropathol Exp Neurol 2022; 82: 21–28.

12.

Agrawal

Farfel

Arfanakis

, et al. Brain autopsies of critically ill COVID-19 patients demonstrate heterogeneous profile of acute vascular injury, inflammation and age-linked chronic brain diseases. Acta Neuropathol Commun 2022; 10: 186.

13.

Wierzba-Bobrowicz

Krajewski

Tarka

, et al. Neuropathological analysis of the brains of fifty-two patients with COVID-19. Folia Neuropathol 2021; 59: 219.

14.

Colombo

Falasca

Marchioni

, et al. Neuropathology and inflammatory cell characterization in 10 autoptic COVID-19 brains. Cells 2021; 10: 2262.

15.

Lengacher

Tomlinson

Jochum

, et al. Neuropathological assessment of the olfactory bulb and tract in individuals with COVID-19. Acta Neuropathol Commun 2024; 12: 70.

16.

Khan

Yoo

Clijsters

, et al. Visualizing in deceased COVID-19 patients how SARS-CoV-2 attacks the respiratory and olfactory mucosae but spares the olfactory bulb. Cell 2021; 184: 5932–5949 e5915.

17.

Thakur

Miller

Glendinning

, et al. COVID-19 neuropathology at Columbia University Irving Medical Center/New York Presbyterian Hospital. Brain 2021; 144: 2696–2708.

18.

Augusto-Oliveira

Arrifano

Lopes-Araujo

, et al.

What do microglia really do in healthy adult brain?

Cells 2019; 8: 1293.

19.

Garwood

Ratcliffe

Simpson

, et al. Review: astrocytes in Alzheimer's disease and other age-associated dementias: a supporting player with a central role. Neuropathol Appl Neurobiol 2017; 43: 281–298.

20.

Zhao

Lukiw

. Ubiquity of the SARS-CoV-2 receptor ACE2 and upregulation in limbic regions of Alzheimer's disease brain. Folia Neuropathol 2021; 59: 232–238.

21.

Krey

Huber

Hoglinger

, et al.

Can SARS-CoV-2 infection lead to neurodegeneration and Parkinson's disease?

Brain Sci 2021; 11: 1654.

22.

Leta

Rodriguez-Violante

Abundes

, et al. Parkinson's disease and post-COVID-19 syndrome: the Parkinson's long-COVID spectrum. Mov Disord 2021; 36: 1287–1289.

23.

Wang

Davis

Volkow

, et al. Association of COVID-19 with new-onset Alzheimer's disease. J Alzheimers Dis 2022; 89: 411–414.

24.

Buchman

Leurgans

Kim

, et al. Alzheimer's disease pathology outside of the cerebrum is related to a higher odds of dementia. J Alzheimers Dis 2023; 96: 563–578.

25.

Wiseman

Murray

Faull

, et al. Aggregate-prone brain regions in Parkinson's disease are rich in unique N-terminus alpha-synuclein conformers with high proteolysis susceptibility. NPJ Parkinsons Dis 2024; 10: 1.

26.

Josephs

Dickson

. TDP-43 in the olfactory bulb in Alzheimer's disease. Neuropathol Appl Neurobiol 2016; 42: 390–393.

27.

Braak

Rub

Gai

, et al. Idiopathic Parkinson's disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen. J Neural Transm (Vienna) 2003; 110: 517–536.

28.

Pandey

Bapat

Abraham

, et al. Long COVID: from olfactory dysfunctions to viral Parkinsonism. World J Otorhinolaryngol Head Neck Surg 2024; 10: 137–147.

29.

Amruta

Chastain

Paz

, et al. SARS-CoV-2 mediated neuroinflammation and the impact of COVID-19 in neurological disorders. Cytokine Growth Factor Rev 2021; 58: 1–15.

30.

Libard

Laurell

Cesarini

, et al. Progression of Alzheimer's disease-related pathology and cell counts in a patient with idiopathic normal pressure hydrocephalus. J Alzheimers Dis 2018; 61: 1451–1462.

31.

Braak

Alafuzoff

Arzberger

, et al. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol 2006; 112: 389–404.

32.

Alafuzoff

Ince

Arzberger

, et al. Staging/typing of Lewy body related alpha-synuclein pathology: a study of the BrainNet Europe Consortium. Acta Neuropathol 2009; 117: 635–652.

33.

Thal

Rub

Orantes

, et al. Phases of A beta-deposition in the human brain and its relevance for the development of AD. Neurology 2002; 58: 1791–1800.

34.

Kovacs

Xie

Lee

, et al. Multisite assessment of aging-related tau astrogliopathy (ARTAG). J Neuropathol Exp Neurol 2017; 76: 605–619.

35.

Josephs

Murray

Whitwell

, et al. Updated TDP-43 in Alzheimer's disease staging scheme. Acta Neuropathol 2016; 131: 571–585.

36.

Alafuzoff

Libard

. Ageing-related neurodegeneration and cognitive decline. Int J Mol Sci 2024; 25: 4065.

37.

Parkkinen

Soininen

Laakso

, et al. Alpha-synuclein pathology is highly dependent on the case selection. Neuropathol Appl Neurobiol 2001; 27: 314–325.

38.

Ubeda-Banon

Saiz-Sanchez

Flores-Cuadrado

, et al. The human olfactory system in two proteinopathies: Alzheimer's and Parkinson's diseases. Transl Neurodegener 2020; 9: 22.

39.

Diez

Ortiz-Teran

TSC

, et al. Tau propagation in the brain olfactory circuits is associated with smell perception changes in aging. Nat Commun 2024; 15: 4809.

40.

Attems

Lintner

Jellinger

. Olfactory involvement in aging and Alzheimer's disease: an autopsy study. J Alzheimers Dis 2005; 7: 149–157; discussion 173–180.

41.

Tsuboi

Wszolek

Graff-Radford

, et al. Tau pathology in the olfactory bulb correlates with Braak stage, Lewy body pathology and apolipoprotein epsilon4. Neuropathol Appl Neurobiol 2003; 29: 503–510.

42.

Yokota

Miki

Nakashima-Yasuda

, et al. Cerebral Braak stage and amygdala granular fuzzy astrocyte status have independent effects on neuronal 3R-tau and 4R-tau accumulations in the olfactory bulb, respectively, in cases with low to intermediate AD neuropathologic change. Acta Neuropathol 2025; 149: 36.

43.

Braak

Del Tredici

Rub

, et al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 2003; 24: 197–211.

44.

Dowd

McKernan

. Back to the future: lessons from past viral infections and the link with Parkinson's disease. Neuronal Signal 2021; 5: NS20200051.

45.

Rahmati

Yon

Lee

, et al. New-onset neurodegenerative diseases as long-term sequelae of SARS-CoV-2 infection: a systematic review and meta-analysis. J Med Virol 2023; 95: e28909.

46.

Kaufer

Schreiber

Hartke

, et al. Microgliosis and neuronal proteinopathy in brain persist beyond viral clearance in SARS-CoV-2 hamster model. EBioMedicine 2022; 79: 103999.

47.

Brundin

Nath

Beckham

. Is COVID-19 a perfect storm for Parkinson's disease? Trends Neurosci 2020; 43: 931–933.

48.

Philippens

Boszormenyi

Wubben

JAM

, et al. Brain inflammation and intracellular alpha-synuclein aggregates in macaques after SARS-CoV-2 infection. Viruses 2022; 14: 776.

49.

Neumann

Sampathu

Kwong

, et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 2006; 314: 130–133.

50.

Josephs

Whitwell

Weigand

, et al. TDP-43 is a key player in the clinical features associated with Alzheimer's disease. Acta Neuropathol 2014; 127: 811–824.

51.

Nelson

Lee

Cykowski

, et al. LATE-NC staging in routine neuropathologic diagnosis: an update. Acta Neuropathol 2023; 145: 159–173.

52.

Nelson

Dickson

Trojanowski

, et al. Limbic-predominant age-related TDP-43 encephalopathy (LATE): consensus working group report. Brain 2019; 142: 1503–1527.

53.

Josephs

Murray

Whitwell

, et al. Staging TDP-43 pathology in Alzheimer's disease. Acta Neuropathol 2014; 127: 441–450.

54.

Takeda

Iijima

Uchihara

, et al. TDP-43 pathology progression along the olfactory pathway as a possible substrate for olfactory impairment in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 2015; 74: 547–556.

55.

Nishihira

Tan

Onodera

, et al. Sporadic amyotrophic lateral sclerosis: two pathological patterns shown by analysis of distribution of TDP-43-immunoreactive neuronal and glial cytoplasmic inclusions. Acta Neuropathol 2008; 116: 169–182.

56.

Yang

Wang

, et al. The SARS-CoV-2 main protease induces neurotoxic TDP-43 cleavage and aggregates. Signal Transduct Target Ther 2023; 8: 109.

57.

Idrees

Kumar

. SARS-CoV-2 spike protein interactions with amyloidogenic proteins: potential clues to neurodegeneration. Biochem Biophys Res Commun 2021; 554: 94–98.

58.

Laudanski

Hajj

Restrepo

, et al. Dynamic changes in central and peripheral neuro-injury vs. Neuroprotective serum markers in COVID-19 are modulated by different types of anti-viral treatments but do not affect the incidence of late and early strokes. Biomedicines 2021; 9: 1791.

59.

Rahic

Buratti

Cappelli

. Reviewing the potential links between viral infections and TDP-43 proteinopathies. Int J Mol Sci 2023; 24: 1581.

60.

Tavassoly

Safavi

Tavassoly

. Seeding brain protein aggregation by SARS-CoV-2 as a possible long-term complication of COVID-19 infection. ACS Chem Neurosci 2020; 11: 3704–3706.

61.

Bhardwaj

Gadhave

Kapuganti

, et al. Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes. Nat Commun 2023; 14: 945.

62.

De Canniere

. The olfactory striae: a historical perspective on the inconsistent anatomy of the bulbar projections. J Anat 2024; 244: 170–183.

63.

Minett

Classey

Matthews

, et al. Microglial immunophenotype in dementia with Alzheimer's pathology. J Neuroinflamm 2016; 13: 135.

64.

Lier

Streit

Bechmann

. Beyond activation: characterizing microglial functional phenotypes. Cells 2021; 10: 2236.

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