Contribution of perforant path synaptic markers and GSK-3 to cognitive impairment and Alzheimeŕs disease

Premi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2017-2018La frecuencia de la enfermedad de Alzheimer (EA) y otras demencias está aumentando en todo el mundo debido al progresivo envejecimiento de la población. A pesar de que hay marcardores patológicos específicos para determinadas patologías, p.e. placas seniles y ovillos neurofibrilares en la EA, todavía se desconoce cuál es la patología subyacente al deterioro cognitivo en sí. Para identificar nuevas dianas terapéuticas es preciso determinar qué factores pueden estar asociados al deterioro cognitivo y posteriormente, desarrollar nuevos modelos experimentales que nos permitan profundizar en su estudio y analizar su posible contribución. En esta tesis, se estudiaron las correlaciones entre marcadores sinápticos (sinaptofisina, proteína sináptica vesicular-2 (SV2) y glutamato vesicular transportador-1 (VGLUT1)) y otros cambios patológicos como la fase de Thal, estadio Braak, enfermedad cerebrovascular, esclerosis hipocampal, y la proteína de respuesta transactiva de unión a ADN de 43kDa (TDP-43) patológica, con el estado cognitivo y la función cognitiva global en individuos mayores de 90 años. Observamos que ninguna de las variables diferenciaba a individuos control de pacientes con deterioro cognitivo-no-demencia, pero todas ellas diferenciaban a controles de pacientes con demencia. Además, sinaptofisina y SV2 estaban aumentadas, mientras que Braak, TDP-43 y esclerosis hipocámpica estaban reducidos en el grupo de individuos con deterioro cognitivo-no-demencia comparado con el grupo demencia; sin embargo, la fase Thal y VGLUT1 no diferenciaban a estos dos grupos. Todas las variables analizadas se asociaban con los tests cognitivos. Finalmente, el análisis simultáneo de todas las variables mostró que sinaptofisina, el estadio Braak, TDP-43 y esclerosis hipocámpica se asociaban con la función cognitiva global. Concluímos que aunque la patología amiloide y los cambios en las sinasis glutamatérgicas podían ser eventos iniciales, en nuestro estudio los cerebros del grupo de deterioro cognitivo no-demencia eran indistinguibles de los cerebros del grupo control; además, la patología neurofibrilar, esclerosis hipocampal, TDP-43 y la pérdida sináptica de la via perforante fueron los mayores contribuidores del deterioro cognitivo en los más mayores. En la segunda parte de esta tesis, tratamos de dilucidar la contribución de la glucógeno sintasa quinasa 3 (GSK-3) en el deterioro cognitivo y patología amiloide en un modelo murino de EA (PDAPP). GSK-3 ha sido ampliamente estudiada en esta enfermedad, pero la contribución individual de cada una de sus dos isoformas, α y β, sobretodo α es desconocida. En este estudio generamos líneas de ratones GSK-3α y GSK-3β knockout condicionados con el sistema CRE-loxP, sin afectación de GSK-3β y GSK-3α respectivamente, pero no logramos generar GSK-3αβ doble knockouts. También generamos triples transgénicos con ratones PDAPP, y observamos que la reducción de GSK-3α reducía significativamente la carga amiloide, los déficits de aprendizaje e hiperactividad de los ratones trasngénicos. Sin embargo, no logramos obtener ratones triples transgénicos con GSK-3β knockout que sobrevivieran más allá de los 6 meses. Concluímos que aquellas estrategias terapéuticas que reduzcan selectivamente una de las dos isoformas posiblemente sean más seguras y bien toleradas que aquellas que inhiban ambas; que un exceso de APP o derivados pueden tener efectos perjudiciales en los ratones GSK-3β knockout condicionados; y que los inhibidores selectivos de GSK-3α pueden ser una buena estrategia terapéutica para la EA. En resumen, los trastornos cognitivos son entidades complejas en las que múltiples factores pueden interactuar y contribuir al deterioro cognitivo. En esta tesis hemos observado que, en humanos, la patología neurofibrilar, esclerosis hipocámpica, la patología TDP-43 y la pérdida sináptica de la vía perforante puede contribuir al deterioro cognitivo en mayores de 90 años. Además, en ratones, la reducción de GSK-3α redujo la carga amiloide y los déficits cognitivos y conductuales, destacando GSK-3α como un posible contriuyente al deterioro cognitivo, y como una posible diana terapéutica en la EA.The frequency of Alzheimer's disease (AD) and other dementias is increasing worldwide due to the progressive ageing of the population. Although distinct pathology is related to specific entities and dementia, such as senile plaques and neurofibrillary tangles in AD, there is still uncertainty about the pathology underlying cognitive impairment itself. To identify new therapeutic targets, we need to determine which factors can be associated to cognitive impairment, and then, develop experimental models to confirm their contribution. In this dissertation, we first studied the correlations between synaptic markers (synaptophysin, synaptic vesicle protein 2 (SV2) and vesicular glutamate transporter 1 (VGLUT1), and other pathological changes such as Thal phase, Braak stage, cerebrovascular disease, hippocampal sclerosis, and pathological 43-kDa transactive response sequence DNA-binding protein (TDP-43), with cognitive status and global cognitive function in the oldest-old. We first observed that neither measure differentiated control from cognitive impairment but no dementia group. Secondly, we found that all measures distinguished dementia from control groups. Third, synaptophysin and SV2 were increased, whereas Braak stage, TDP-43 and hippocampal sclerosis were reduced in the cognitive impairment but no dementia group compared to the dementia group, while Thal phase and VGLUT1 did not distinguish between these two groups. Fourth, all markers were associated with cognitive test scores. And fifth, when all measures were analyzed simultaneously, a reduction in synaptophysin, a high Braak stage and the presence of pathological TDP-43 and hippocampal sclerosis were associated with a worse global cognitive function. We concluded that although changes in plaque pathology and glutamatergic synapses may be early events, in our study and with our measures, cognitive impairment but no dementia group brains were indistinguishable from control brains; and moreover, that tangle pathology, hippocampal sclerosis, TDP-43 pathology and perforant pathway synaptic loss were the major contributors to global cognitive function in the oldest-old. In the second part of this dissertation, we sought to elucidate the contribution of Glycogen synhase kinase 3 (GSK-3) in cognitive impairment and AD pathology in a mouse model of AD (PDAPP). GSK-3 has been widely studied in AD, and it has two isoforms, α and β, but the individual contribution of each isoform, mainly GSK-3α, remain poorly understood. In this study, we generated GSK-3 α and β conditional knockout lines using CRE-loxP system, without targeting GSK-3β and GSK-3α respectively, but failed to generate the double GSK-3αβ knockout. We generated triple transgenic lines with PDAPP mice, and we found that GSK-3α knockdown showed a significant effect on reducing senile plaques, learning deficits and hyperactivity in transgenic mice. Interestingly, we were unable to obtain triple conditional GSK-3β knockout that survived beyond 6 months. We concluded that strategies selectively reducing one isoform of GSK-3 may be safer and well tolerated than GSK-3 inhibitors targeting both; that an excess of APP or derivatives have deleterious effects in GSK-3β conditional knockout mice; and that developing isoform specific GSK-3α inhibitors can be a good therapeutic strategy for AD. In summary, cognitive disorders are complex entities in which multiple factors may interact and contribute to the cognitive impairment. In this dissertation we observed that, in humans, tangle pathology, hippocampal sclerosis, TDP-43 pathology and perforant pathway synaptic loss may contribute to cognitive impairment in the oldest-old. Additionally, in mice, GSK-3α knockout reduced plaque load, and behavioral and cognitive impairment, highlighting GSK-3α as a possible contributor to cognitive impairment, and as a potential therapeutic target in Alzheimer's disease

Teaching case 3-2019: Are nuclear clefts or invaginations the niche of intranuclear inclusions in FTLD-TDP?

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Cognitive decline in amyotrophic lateral sclerosis: Neuropathological substrate and genetic determinants

Proteïna TDP-43; Esclerosi lateral amiotròfica; Demència frontotemporalProteína TDP-43; Esclerosis lateral amiotrófica; Demencia frontotemporalTDP-43 protein; Amyotrophic lateral sclerosis; Frontotemporal dementiaCognitive impairment and behavioral changes in amyotrophic lateral sclerosis (ALS) are now recognized as part of the disease. Whether it is solely related to the extent of TDP-43 pathology is currently unclear. We aim to evaluate the influence of age, genetics, neuropathological features, and concomitant pathologies on cognitive impairment in ALS patients. We analyzed a postmortem series of 104 ALS patients and retrospectively reviewed clinical and neuropathological data. We assessed the burden and extent of concomitant pathologies, the role of APOE ε4 and mutations, and correlated these findings with cognitive status. We performed a logistic regression model to identify which pathologies are related to cognitive impairment. Cognitive decline was recorded in 38.5% of the subjects. Neuropathological features of frontotemporal lobar degeneration (FTLD) were found in 32.7%, explaining most, but not all, cases with cognitive impairment. Extent of TDP-43 pathology and the presence of hippocampal sclerosis were associated with cognitive impairment. Mutation carriers presented a higher burden of TDP-43 pathology and FTLD more frequently than sporadic cases. Most cases (89.4%) presented some degree of concomitant pathologies. The presence of concomitant pathologies was associated with older age at death. FTLD, but also Alzheimer’s disease, were the predominant underlying pathologies explaining the cognitive impairment in ALS patients. In sum, FTLD explained the presence of cognitive decline in most but not all ALS cases, while other non-FTLD related findings can influence the cognitive status, particularly in older age groups.SBE is a recipient of the Rio-Hortega post-residency grant from the Instituto de Salud Carlos III, Spain. This study was partially funded by Fundació Marató de TV3 (grant no. 20141610 to EG and no. 20143710 to RRG) and Fondo Europeo de Desarrollo Regional (FEDER) (PI15/01618 to RRG). AA is funded by Departament de Salut de la Generalitat de Catalunya, Pla estratègic de recerca i innovació en salut (PERIS) 2016–2020 (SLT002/16/00329). JG is recipient of the Instituto de Salud Carlos III-FEDER grants (PI16/01673 and PI19/00593

Wasteosomes (corpora amylacea) as a hallmark of chronic glymphatic insufficiency

In different organs and tissues, the lymphatic system serves as a drainage system for interstitial fluid and is useful for removing substances that would otherwise accumulate in the interstitium. In the brain, which lacks lymphatic circulation, the drainage and cleaning function is performed by the glymphatic system, called so for its dependence on glial cells and its similar function to that of the lymphatic system. In the present article, we define glymphatic insufficiency as the inability of the glymphatic system to properly perform the brain cleaning function. Furthermore, we propose that corpora amylacea or wasteosomes, which are protective structures that act as waste containers and accumulate waste products, are, in fact, a manifestation of chronic glymphatic insufficiency. Assuming this premise, we provide an explanation that coherently links the formation, distribution, structure, and function of these bodies in the human brain. Moreover, we open up new perspectives in the study of the glymphatic system since wasteosomes can provide information about which variables have the greatest impact on the glymphatic system and which diseases occur with chronic glymphatic insufficiency. For example, based on the presence of wasteosomes, it seems that aging, sleep disorders, and cerebrovascular pathologies have the highest impact on the glymphatic system, whereas neurodegenerative diseases have a more limited impact. Furthermore, as glymphatic insufficiency is a risk factor for neurodegenerative diseases, information provided by wasteosomes could help to define the strategies and actions that can prevent glymphatic disruptions, thus limiting the risk of developing neurodegenerative diseases

Uncovering tau in wasteosomes (corpora amylacea) of Alzheimer's disease patients

Brain corpora amylacea, recently renamed as wasteosomes, are polyglucosan bodies that appear during aging and some neurodegenerative conditions. They collect waste substances and are part of a brain cleaning mechanism. For decades, studies on their composition have produced inconsistent results and the presence of tau protein in them has been controversial. In this work, we reanalyzed the presence of this protein in wasteosomes and we pointed out a methodological problem when immunolabeling. It is well known that to detect tau it is necessary to perform an antigen retrieval. However, in the case of wasteosomes, an excessive antigen retrieval with boiling dissolves their polyglucosan structure, releases the entrapped proteins and, thus, prevents their detection. After performing an adequate pre-treatment, with an intermediate time of boiling, we observed that some brain wasteosomes from patients with Alzheimer's disease (AD) contained tau, while we did not detect tau protein in those from non-AD patients. These observations pointed the different composition of wasteosomes depending on the neuropathological condition and reinforce the role of wasteosomes as waste containers

Corpora Amylacea in the Human Brain Exhibit Neoepitopes of a Carbohydrate Nature

Corpora amylacea (CA) in the human brain are polyglucosan bodies that accumulate residual substances originated from aging and both neurodegenerative and infectious processes. These structures, which act as waste containers, are released from the brain to the cerebrospinal fluid, reach the cervical lymph nodes via the meningeal lymphatic system and may be phagocytosed by macrophages. Recent studies indicate that CA present certain neoepitopes (NEs) that can be recognized by natural antibodies of the IgM class, and although evidence of different kinds suggests that these NEs may be formed by carbohydrate structures, their precise nature is unknown. Here, we adapted standard techniques to examine this question. We observed that the preadsorption of IgMs with specific carbohydrates has inhibitory effects on the interaction between IgMs and CA, and found that the digestion of CA proteins had no effect on this interaction. These findings point to the carbohydrate nature of the NEs located in CA. Moreover, the present study indicates that, in vitro, the binding between certain natural IgMs and certain epitopes may be disrupted by certain monosaccharides. We wonder, therefore, whether these inhibitions may also occur in vivo. Further studies should now be carried out to assess the possible in vivo effect of glycemia on the reactivity of natural IgMs and, by extension, on natural immunity

Uncovering tau in wasteosomes (corpora amylacea) of Alzheimer’s disease patients

Brain corpora amylacea, recently renamed as wasteosomes, are polyglucosan bodies that appear during aging and some neurodegenerative conditions. They collect waste substances and are part of a brain cleaning mechanism. For decades, studies on their composition have produced inconsistent results and the presence of tau protein in them has been controversial. In this work, we reanalyzed the presence of this protein in wasteosomes and we pointed out a methodological problem when immunolabeling. It is well known that to detect tau it is necessary to perform an antigen retrieval. However, in the case of wasteosomes, an excessive antigen retrieval with boiling dissolves their polyglucosan structure, releases the entrapped proteins and, thus, prevents their detection. After performing an adequate pre-treatment, with an intermediate time of boiling, we observed that some brain wasteosomes from patients with Alzheimer’s disease (AD) contained tau, while we did not detect tau protein in those from non-AD patients. These observations pointed the different composition of wasteosomes depending on the neuropathological condition and reinforce the role of wasteosomes as waste containers

Corpora amylacea act as containers that remove waste products from the brain

Corpora amylacea (CA) in the human brain are granular bodies formed by polyglucosan aggregates that amass waste products of different origins. They are generated by astrocytes, mainly during aging and neurodegenerative conditions, and are located predominantly in periventricular and subpial regions. This study shows that CA are released from these regions to the cerebrospinal fluid and are present in the cervical lymph nodes, into which cerebrospinal fluid drains through the meningeal lymphatic system. We also show that CA can be phagocytosed by macrophages. We conclude that CA can act as containers that remove waste products from the brain and may be involved in a mechanism that cleans the brain. Moreover, we postulate that CA may contribute in some autoimmune brain diseases, exporting brain substances that interact with the immune system, and hypothesize that CA may contain brain markers that may aid in the diagnosis of certain brain diseases

Cognitive decline in amyotrophic lateral sclerosis: Neuropathological substrate and genetic determinants

Cognitive impairment and behavioral changes in amyotrophic lateral sclerosis (ALS) are now recognized as part of the disease. Whether it is solely related to the extent of TDP-43 pathology is currently unclear. We aim to evaluate the influence of age, genetics, neuropathological features, and concomitant pathologies on cognitive impairment in ALS patients. We analyzed a postmortem series of 104 ALS patients and retrospectively reviewed clinical and neuropathological data. We assessed the burden and extent of concomitant pathologies, the role of APOE ε4 and mutations, and correlated these findings with cognitive status. We performed a logistic regression model to identify which pathologies are related to cognitive impairment. Cognitive decline was recorded in 38.5% of the subjects. Neuropathological features of frontotemporal lobar degeneration (FTLD) were found in 32.7%, explaining most, but not all, cases with cognitive impairment. Extent of TDP-43 pathology and the presence of hippocampal sclerosis were associated with cognitive impairment. Mutation carriers presented a higher burden of TDP-43 pathology and FTLD more frequently than sporadic cases. Most cases (89.4%) presented some degree of concomitant pathologies. The presence of concomitant pathologies was associated with older age at death. FTLD, but also Alzheimer's disease, were the predominant underlying pathologies explaining the cognitive impairment in ALS patients. In sum, FTLD explained the presence of cognitive decline in most but not all ALS cases, while other non-FTLD related findings can influence the cognitive status, particularly in older age groups

R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils

Heterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1–2% of all cases of Parkinson’s disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2—either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10(Thr73)) was measured by quantitative multiplexed immunoblotting for pRab10(Thr73)/total Rab10 as well as targeted mass-spectrometry for absolute pRab10(Thr73) occupancy. We found a significant over fourfold increase in pRab10(Thr73) phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10(Thr73) phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10(Thr73) phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00401-021-02325-z