Martí Colom-Cadena

Confluence of α-synuclein, tau, and β-amyloid pathologies in dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is pathologically characterized by α-synuclein aggregates in the brain. Most patients with DLB also show cerebral Alzheimer disease-type pathology (i.e. β-amyloid plaques and hyperphosphorylated tau deposits). It is unclear whether this overlap is coincidental or driven by specific regional or cellular interactions. The aims of this study were to investigate the regional convergence of α-synuclein, tau, and β-amyloid and to identify patterns of cellular co-occurrence of tau and α-synuclein in DLB. The study group consisted of 22 patients who met clinical and neuropathologic criteria for DLB. Protein aggregates were assessed semiquantitatively in 17 brain areas. APOE and MAPT genotypes were determined. Cellular co-occurrence of tau and α-synuclein was evaluated by double immunofluorescence. We found that total β-amyloid pathology scores correlated positively with total α-synuclein pathology scores (ρ = 0.692, p = 0.001). The factors that correlated best with the amount of α-synuclein pathology were the severity of β-amyloid pathology and presence of the MAPT H1 haplotype. Tau and α-synuclein frequently colocalized in limbic areas, but no correlation between total pathology scores was observed. This study confirms and extends the role of β-amyloid deposition and the MAPT H1 haplotype as contributing factors in DLB pathogenesis and demonstrates the confluence of multiple agents in neurodegenerative diseases.

MAPT H1 haplotype is associated with enhanced α-synuclein deposition in dementia with Lewy bodies

The microtubule-associated protein tau (MAPT) H1 haplotype has been identified as a genetic risk factor for synucleinopathies. However, whether it modulates tau or α-synuclein pathology remains unknown. Our aim was to investigate the relationship between MAPT haplotypes and pathologic aggregates of tau and α-synuclein in pathologically confirmed cases of dementia with Lewy bodies (DLB). Twenty-two cases fulfilling clinical and neuropathological criteria for DLB were included. Clinical and neuropathological data were collected, and APOE and MAPT genotypes were determined. Tau and α-synuclein pathology was assessed semiquantitatively in 17 brain areas and total scores were calculated. DLB H1/H1 (n = 12) and H2 carriers (n = 10) did not differ in demographics, clinical variables, concomitant Alzheimers pathology, or APOE genotype. Total α-synuclein scores were significantly increased in the H1/H1 group (p = 0.011), largely due to an increase in brainstem regions. This difference was driven by an increase in Lewy bodies and diffuse and punctuate cytoplasmatic α-synuclein aggregates (p = 0.007 and p = 0.025 respectively). These findings provide a mechanistic link for the genetic association between MAPT haplotypes and synucleinopathies.

Autosomal-dominant Alzheimer's disease mutations at the same codon of amyloid precursor protein differentially alter Aβ production

Autosomal‐dominant Alzheimers disease (ADAD) is a genetic disorder caused by mutations in Amyloid Precursor Protein (APP) or Presenilin (PSEN) genes. Studying the mechanisms underlying these mutations can provide insight into the pathways that lead to AD pathology. The majority of biochemical studies on APP mutations to‐date have focused on comparing mechanisms between mutations at different codons. It has been assumed that amino acid position is a major determinant of protein dysfunction and clinical phenotype. However, the differential effect of mutations at the same codon has not been sufficiently addressed. In the present study we compared the effects of the aggressive ADAD‐associated APP I716F mutation with I716V and I716T on APP processing in human neuroglioma and CHO‐K1 cells. All APP I716 mutations increased the ratio of Aβ42/40 and changed the product line preference of γ‐secretase towards Aβ38 production. In addition, the APP I716F mutation impaired the ε‐cleavage and the fourth cleavage of γ‐secretase and led to abnormal APP β‐CTF accumulation at the plasma membrane. Taken together, these data indicate that APP mutations at the same codon can induce diverse abnormalities in APP processing, some resembling PSEN1 mutations. These differential effects could explain the clinical differences observed among ADAD patients bearing different APP mutations at the same position.

Non-fibrillar oligomeric amyloid-β within synapses

Alzheimers disease (AD) is characterized by memory loss, insidious cognitive decline, profound neurodegeneration, and the extracellular accumulation of amyloid-β (Aβ) peptide in senile plaques and intracellular accumulation of tau in neurofibrillary tangles. Loss and dysfunction of synapses are believed to underlie the devastating cognitive decline in AD. A large amount of evidence suggests that oligomeric forms of Aβ associated with senile plaques are toxic to synapses, but the precise sub-synaptic localization of Aβ and which forms are synaptotoxic remain unknown. Here, we characterize the sub-synaptic localization of Aβ oligomers using three high-resolution imaging techniques, stochastic optical reconstruction microscopy, immunogold electron microscopy, and Förster resonance energy transfer in a plaque-bearing mouse model of AD. With all three techniques, we observe oligomeric Aβ inside synaptic terminals. Further, we tested a panel of Aβ antibodies using the relatively high-throughput array tomography technique to determine which forms are present in synapses. Our results show that different oligomeric Aβ species are present in synapses and highlight the potential of array tomography for rapid testing of aggregation state specific Aβ antibodies in brain tissue.

Synaptic phosphorylated α-synuclein in dementia with Lewy bodies

Dementia with Lewy bodies is characterized by the accumulation of Lewy bodies and Lewy neurites in the CNS, both of which are composed mainly of aggregated α-synuclein phosphorylated at Ser129. Although phosphorylated α-synuclein is believed to exert toxic effects at the synapse in dementia with Lewy bodies and other α-synucleinopathies, direct evidence for the precise synaptic localization has been difficult to achieve due to the lack of adequate optical microscopic resolution to study human synapses. In the present study we applied array tomography, a microscopy technique that combines ultrathin sectioning of tissue with immunofluorescence allowing precise identification of small structures, to quantitatively investigate the synaptic phosphorylated α-synuclein pathology in dementia with Lewy bodies. We performed array tomography on human brain samples from five patients with dementia with Lewy bodies, five patients with Alzheimers disease and five healthy control subjects to analyse the presence of phosphorylated α-synuclein immunoreactivity at the synapse and their relationship with synapse size. Main analyses were performed in blocks from cingulate cortex and confirmed in blocks from the striatum of cases with dementia with Lewy bodies. A total of 1 318 700 single pre- or postsynaptic terminals were analysed. We found that phosphorylated α-synuclein is present exclusively in dementia with Lewy bodies cases, where it can be identified in the form of Lewy bodies, Lewy neurites and small aggregates (<0.16 µm3). Between 19% and 25% of phosphorylated α-synuclein deposits were found in presynaptic terminals mainly in the form of small aggregates. Synaptic terminals that co-localized with small aggregates of phosphorylated α-synuclein were significantly larger than those that did not. Finally, a gradient of phosphorylated α-synuclein aggregation in synapses (pre > pre + post > postsynaptic) was observed. These results indicate that phosphorylated α-synuclein is found at the presynaptic terminals of dementia with Lewy bodies cases mainly in the form of small phosphorylated α-synuclein aggregates that are associated with changes in synaptic morphology. Overall, our data support the notion that pathological phosphorylated α-synuclein may disrupt the structure and function of the synapse in dementia with Lewy bodies.

Regional Overlap of Pathologies in Lewy Body Disorders.

Lewy body disorders (LBD) are common neurodegenerative diseases characterized by the presence of aggregated α-synuclein in Lewy bodies and Lewy neurites in the central and peripheral nervous systems. The brains of patients with LBD often display other comorbid pathologies, i.e. insoluble tau, β-amyloid aggregates, TAR DNA-binding protein 43 (TDP-43) deposits, and argyrophilic grain disease (AGD). The incidence and physiological relevance of these concurrent pathological findings remain controversial. We performed a semiquantitative detailed mapping of α-synuclein, tau, β-amyloid (Aβ), TDP-43, and AGD pathologies in 17 areas in 63 LBD cases (44 with Parkinson disease [PD], 28 with dementia, and 19 with dementia with Lewy bodies). APOE and MAPT genetic variants were also investigated. A majority of LBD cases had 2 or 3 concomitant findings, particularly Alzheimer disease-related pathology. Pathological stages of tau, β-amyloid and α-synuclein pathologies were increased in cases with dementia. Aβ score was the best correlate of the time to dementia in PD. In addition, β-amyloid deposition correlated with α-synuclein load in all groups. MAPT H1 haplotype did not influence any assessed pathology in PD. These results highlight the common concurrence of pathologies in patients with LBD that may have an impact on the clinical expression of the diseases.

YKL-40 (Chitinase 3-like I) is expressed in a subset of astrocytes in Alzheimer’s disease and other tauopathies

BackgroundThe innate immune system is known to be involved early in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative disorders. The inflammatory response in the central nervous system can be measured postmortem or through a series of inflammatory mediator surrogates. YKL-40 (also named Chitinase-3-like I) has been frequently investigated in body fluids as a surrogate marker of neuroinflammation in AD and other neurological disorders. However, the expression pattern of YKL-40 in the human brain with neurodegenerative pathology remains poorly investigated. Our aim was to study the cellular expression pattern of YKL-40 in the brain of patients with clinical and neuropathological criteria for AD (n = 11); three non-AD tauopathies: Pick’s disease (PiD; n = 8), corticobasal degeneration (CBD; n = 8) and progressive supranuclear palsy (PSP; n = 9) and a group of neurologically healthy controls (n = 6).MethodsSemiquantitative neuropathological evaluation and quantitative confocal triple immunofluorescence studies were performed. An in-house algorithm was used to detect and quantify pathology burden of random regions of interest on a full tissue-section scan. Kruskal-Wallis and Dunn’s multiple comparison tests were performed for colocalization and quantification analyses.ResultsWe found that brain YKL-40 immunoreactivity was observed in a subset of astrocytes in all four diseases and in controls. There was a strong colocalization between YKL-40 and the astroglial marker GFAP but not with neuronal nor microglial markers. Intriguingly, YKL-40-positive astrocytes were tau-negative in PSP, CBD and PiD. The number of YKL-40-positive astrocytes was increased in tauopathies compared with that in controls. A positive correlation was found between YKL-40 and tau immunoreactivities.ConclusionsThis study confirms that YKL-40 is expressed by a subset of astrocytes in AD and other tauopathies. YKL-40 expression is elevated in several neurodegenerative conditions and correlates with tau pathology.

Pick's pathology in Parkinson's disease with dementia

In Parkinson’s disease (PD) dementia occurs in up to 40% of patients. Its neuropathological substrate is still controversial. We report clinical and neuropathological findings in a patient with PD who developed dementia during the course of the disease. We show that in PD, Pick’s pathology may also contribute to cognitive decline. The patient was a 62-year-old woman presenting with right hand resting tremor, with good levodopa response. Two years later, she suffered from depressive symptoms and episodes of disorientation. Visual hallucinations, rapid eye movement (REM) sleep behaviour disorder and loss of memory appeared 4 years after disease onset. At this point neurological evaluation showed a predominant right-sided mixed parkinsonism, in a Hoehn & Yahr stage II. Orthostatic hypotension was also observed. Brain MRI showed isolated lacunes in basal ganglia. In EEG, slow base rhythm appeared. Mini Mental State Examination (MMSE) was 26/30. Neuropsychological evaluation disclosed severe impairment of long-term retention and verbal working memory and mild impairment of semantic verbal fluency, visual gnosis and sequencing capacity, overall suggesting corticosubcortical impairment. Cognitive decline worsened progressively after 8 years of disease onset and she presented fluctuating confusional states, auditory hallucinations, compulsive eating, nocturnal restlessness and prominent disinhibition. Gate became worse, mild dyskinesias appeared and falls became frequent. She complained of painful dystonia in her legs and place reduplication delusions. At that time, MMSE was 21/30 and galantamine was started. She deteriorated rapidly and died due to pneumonia at the age of 77 after 15 years of disease duration. Brain was donated at the Neurological Tissue Bank of the Biobanc-Hospital Clinic-IDIBAPS after obtaining written informed consent for its use for diagnostic and research purposes. Fresh brain weight was 1135 g. Macroscopically, mild diffuse oedema with widening of gyral pattern and collapsed sulci was observed. Coronal sections showed mild ventricular enlargement and moderate atrophy of posterior hippocampus. Marked pallor of substantia nigra (SN) and locus ceruleus (LC) was detected. Five-micrometre-thick sections were obtained from each brain area (Table 1) and stained with haematoxylin-eosin (HE) and by immunohistochemistry using the following antibodies: anti-bA4-amyloid (clone 6F/3D, DAKO, Glostrup, Denmark), anti-phosphorylated tau (clone AT8; Thermo Scientific, Rockford, IL, USA), anti-phospho-tau ser396 (AB557815, polyclonal; Calbiochem, La Jolla, San Diego, CA, USA), anti-ubiquitin (DAKO), anti-a-synuclein (clone KM51; Novocastra, Newcastle, UK), anti-asynuclein (AB5038, polyclonal; Calbiochem), antiphospho-a-synuclein Ser129 (clone pSyn#64; Wako, Richmond, VA, USA), anti-b-synuclein (ab6165, polyclonal; Abcam, Cambridge, UK), anti-TDP-43 (clone 2E2-D3; Abnova, Taipei, Taiwan), neurofilaments (clone RT97; Novocastra), anti-RD3 (clone 8E6/C11; Millipore, Temecula, CA, USA), anti-RD4 (clone 1E1/A6; Millipore), anti-a-internexin (clone 2E3; Invitrogen, Carlsbad, CA, USA), anti-a-B-crystallin (clone G2JF; Novocastra). Double immunofluorescence was performed in hippocampus including parahippocampal gyrus, amygdala and frontal cortex combining Tau-AT8 and polyclonal a-synuclein, phospho-tau ser396 and monoclonal phospho-a-synuclein, as well as Tau-RD3 and polyclonal a-synuclein. Sections were incubated with Alexa Fluor 488 or 555-labelled secondary antibodies (Invitrogen). Nuclei were stained with DAPI in Vectashield mount medium (Vector Laboratories, USA). Fluorescence was analysed with Leica inverted fluorescent confocal microscope (Leica TCD SP5-AOBS, Wetzlar, Germany). Microscopically, moderate neuronal loss and gliosis were observed in cortical areas with mild superficial spongiosis in frontolateral and orbitofrontal cortices and cingulum (Figure 1B1). Additionally, frequent ballooned cells were detected in these areas (Figure 1B2). Here, frequent spherical basophilic cytoplasmic neuronal inclusions suggestive of Pick bodies were visible on HE-stained sections, in granular neurons of dentate gyrus of the hippocampus and in parahippocampal gyrus (Figure 1B3). Furthermore, severe loss of pigmented neurons of SN pars compacta and moderate loss of LC neurons was detected. Lewy bodies (LBs) and pale bodies were already visible in HE

Tauopathy with Hippocampal 4-Repeat Tau Immunoreactive Spherical Inclusions in a Patient with PSP: Letter to the Editor

We read with interest the manuscript by Kovacs et al entitled “Tauopathy with hippocampal 4-repeat tau immunoreactive spherical inclusions: a report of three cases” (2). The authors described the clinical and neuropathologic features of three patients with a novel 4-repeat tauopathy characterized by hippocampal spherical inclusions. Previously, Miki and colleagues, also reported another case with similar neuropathologic findings (3). One of the cases reported by Kovacs et al was identified after screening 240 Alzheimer disease (AD) or primary age-related tauopathy (PART) cases, while two of them were identified among 50 patients with subcortical tau pathology consistent with progressive supranuclear palsy (PSP). This prompted us to systematically assess hippocampal sections stained by AT8 immunohistochemistry of 70 PSP and 30 Cortico-Basal Degeneration (CBD) archival cases with the aim to identify potential subjects with hippocampal spherical inclusions. We detected a single case among the PSP group with the same neuropathological features as described by Miki et al and Kovacs et al. The patient was a 69-year-old man who suffered from progressive memory dysfunction, depression, and apathy starting at age of 58. He scored 24/30 at the Mini mental state examination when he was 64. During disease evolution, he developed hallucinations and a rapidly progressive parkinsonism. No further clinical details were available. The clinical diagnosis was dementia with Lewy bodies. The patient died at the age of 69 years after total disease duration of 11 years. There was no family history of neurological disorders or dementia. No mutations in exons 1, 9, 10, 11, 12, and 13 of the MAPT gene were found. Brain was removed after death for diagnostic and research purposes after obtaining written informed consent form the next of kin. Unfixed brain weight was 1115 g. Gross examination revealed mild global brain atrophy accentuated in the hippocampus. The subthalamic nucleus was atrophic and the substantia nigra showed severe pallor. Histological examination confirmed a severe loss of pigmented neurons of the substantia nigra pars compacta, a mild neuronal loss in locus coeruleus and of the external and internal globus pallidus. There was also prominent neuronal loss of the subthalamic nucleus. All these areas were accompanied by astrogliosis and microglial activation. Residual neurons of the brainstem, subthalamic nucleus, and large cholinergic neurons of basal ganglia showed frequent globose tangles. The most striking feature was the severe involvement of the hippocampus. There was a nearly complete loss of pyramidal neurons of the CA4-CA1 sectors and a prominent depletion of granular neurons of the dentate gyrus, consistent with hippocampal sclerosis (Figure 1A,B). Frequent basophilic spherical inclusions of the size of the nucleus reminiscent of Pick bodies were detected in granular neurons of the dentate gyrus and pyramidal neurons of CA1 sector (Figure 1C). Immunohistochemistry showed extensive neuronal and glial ptau pathology. There were frequent tufted astrocytes in all cortical areas, basal ganglia, limbic system, and brainstem nuclei as well as frequent coiled bodies in white matter oligodendrocytes (Figure 1K,L). In addition, a moderate amount of neurofibrillary tangles of the globose type were observed in basal ganglia, subthalamic nucleus, nucleus basalis of Meynert, and brainstem nuclei (Figure 1M). There was also moderate involvement of the pontine nuclei and of the dentate nucleus of the cerebellum, as well as of the spinal cord. The hippocampus showed very frequent spherical inclusions in the dentate gyrus and also in the different sectors of the hippocampus, parahippocampal gyrus, and temporo-occipital cortex together with Neurofibrillary Tangle (NFT) (Figure 1E). Spherical inclusions were also detected in cortical areas and basal ganglia. This pathology was predominantly immunoreactive for 4R tau isoforms and was negative for 3R tau isoforms, except for few NFT in the CA1 sector of the hippocampus (Figure 1F) and an unusual granular cytoplasmic immunoreactivity of some hippocampal neurons (Figure 1F lower right panel). Spherical inclusions were strongly stained by phospho-specific tau antibodies to serine 396, serine 422, threonine 181, and to a lesser extent to serine 262 (all from Calbiochem, La Jolla, CA, USA) (Figure 1G–J). There were only mild B4 amyloid deposits in cortical areas and the limbic system with only few deposits in basal ganglia corresponding to Thal phase 3, and few neuritic plaques (CERAD plaques score A). No a-synuclein or TDP-43 proteins aggregates were detected. These features were consistent with those of PSP with an atypical and severe involvement of the hippocampus, concordant with the observations made by Miki et al and Kovacs and colleagues. Our findings reinforce the notion that this morphological phenotype seems to be different from other established tauopathies. Indeed, our patient presented more prominent supratentorial pathology than in the brainstem in contrast to what is observed in typical PSP (1). The prominent hippocampal involvement is reminiscent of Pick’s disease but instead of 3R tau pathology these cases are dominated by 4R tau isoforms. In accordance with the clinical features described by Kovacs, our patient also presented cognitive impairment that could be related to the severe degeneration of hippocampal structures. Cognitive impairment was not reported in the patient described by Miki et al. Our patient also presented psychiatric symptoms, specifically depression and hallucinations. Although no psychiatric symptoms were reported in the patients described by Kovacs, depression was the main symptom in the case reported by Miki and colleagues.

A CEREBROSPINAL FLUID PANEL OF SYNAPTIC PROTEINS ACROSS THE ENTIRE ALZHEIMER’S DISEASE CONTINUUM

and highest values. Remarkably, CECs CSF for N9 microglia and for J774 macrophages were nearly identical (R1⁄40.94; P<0.0001). CECs CSF for SH-SY5Y, A172 and N9 cells were correlated with one another less (R1⁄40.66-0.78; P<0.0001). CECs CSF for SH-SY5Y, A172 and N9 were strongly correlated with CSF apo A-I (R1⁄40.42-0.7) and apo J (R1⁄40.73-0.85) and weakly correlated with apo E (R1⁄40.28-0.39). After adjustment for apo J, the association between CECs CSF and apo A-I was no longer significant. Conclusions: CECs CSF for SH-SY5Y, A172 and N9 cells exhibit notable variability from individual to individual and from one and another and are determined mainly by CSF apo J levels.