5 research outputs found
Volumetric Manganese Enhanced Magnetic Resonance Imaging in mice (mus musculus)
The present doctoral thesis introduces a method for semi-automatic volumetric analysis of the hippocampus and other distinct brain regions in laboratory mice. The method of volumetric manganese enhanced magnetic resonance imaging (vMEMRI) makes use of the paramagnetic property of the manganese ion, Mn2+, which results in a positive contrast enhancement of specific brain areas on the MR image and enables a more detailed image of brain morphology. The chemical similarity of Mn2+ to Calcium leads to an accumulation of Mn2+ in excited cells and consequentially an enhanced signal in certain brain regions in an activity dependent manner. However, one major drawback for vMEMRI is the toxicity of Mn2+. Therefore, the aims of the thesis have been:
(1) Establishment of a MEMRI protocol in mice
(2) Optimization of a Mn2+ application procedure to reduce toxic side effects
(3) Development of an automatized method to determine hippocampal volume
(4) Validation of vMEMRI analysis
(5) Application of volumetric analysis in mouse models of psychopathology
This thesis splits into 3 studies. Study 1 deals with Mn2+ toxicity and introduces an application method that considerably reduces the toxic side effects of Mn2+. Study 2 validates vMEMRI as a method to reliably determine hippocampal volume and explores its utilization it in animals with genetically and chemically modified hippocampi. Study 3 displays the application vMEMRI in a mouse model of a psychiatric disorder.
Study 1 shows that a single application of Mn2+ in dosages used in current MEMRI studies leads to considerable toxic side effects measurable with physiological, behavioral and endocrine markers. In contrast, a fractionated application of a low dose of Mn2+ is proposed as an alternative to a single injection of a high dose. Repeated application of low dosages of 30 mg/kg Mn2+ showed less toxic side effects compared to the application schemes with higher dosages of 60 mg/kg. Additionally, the best vMEMRI signal contrast was seen for an injection protocol of 30 mg/kg 8 times with an inter-injection interval of 24 h (8x30/24 protocol).
The impact of the 8x30/24 application protocol on longitudinal studies was tested by determining whether learning processes are disturbed. Mice were injected with the 8x30/24 protocol 2 weeks prior to receiving a single footshock. Manganese injected mice showed less contextual freezing to the shock context and a shock context reminder one month after shock application. Furthermore, mice showed increased hyperarousal and no avoidance of shock context related odors. This impairment in fear conditioning indicates a disturbed associative learning of Mn2+ injected mice. Therefore, it was investigated whether Mn2+ application shows a specific disturbance of hippocampus dependent learning. Mice were subjected to habitual and spatial learning protocols 12 h after each injection in a water cross-maze. There was no impairment in learning protocols which allowed for hippocampus-independent habitual learning. However, Mn2+ injected mice were specifically impaired in the hippocampus-dependent spatial learning protocol. Furthermore, it was shown that only mice with higher Mn2+ accumulation showed this impairment. Altogether, the results of this chapter argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects. However, the treatment procedure has to be further improved to allow for an analysis of hippocampus-dependent learning processes as well. Because of the potential side effects, the vMEMRI method was applied as a final experiment in study 2 and 3.
Study 2 introduces the method of vMEMRI, which allows, for the first time, an in vivo semi-automatic detection of hippocampal volume. Hippocampal volume of mice with genetically altered adult neurogenesis and those with chemically lesioned hippocampi could be analyzed with vMEMRI. Even the highly variable differences in hippocampal volume of these animals could be detected with vMEMRI. vMEMRI data correlated with manually obtained volumes and are in agreement with previously reported histological findings, indicating the high reliability of this method.
Study 3 investigates the ability of vMEMRI to detect even small differences in brain morphology by examining volumetric changes of the hippocampus and other brain structures in a mouse model of PTSD supplemented with enriched housing conditions. It was shown, that exposure to a brief inescapable foot shock led to a volume reduction in both the left hippocampus and right central amygdala two months later. Enriched housing decreased the intensity of trauma-associated contextual fear independently of whether it was provided before or after the shock. vMEMRI analysis revealed that enriched housing led to an increase in whole brain volume, including the lateral ventricles and the hippocampus. Furthermore, the enhancement of hippocampal volume through enriched housing was accompanied by the amelioration of trauma-associated PTSD-like symptoms. Hippocampal volume gain and loss was mirrored by ex vivo ultramicroscopic measurements of the hippocampus. Together, these data demonstrate that vMEMRI is able to detect small changes in hippocampal and central amygdalar volumes induced by a traumatic experience in mice.
In conclusion, vMEMRI proves to be very reliable and able to detect small volumetric differences in various brain regions in living mice. vMEMRI opens up a great number possibilities for future research determining neuroanatomical structure, volumes and activity in vivo as well as the ability to repeatedly determine such characteristics within each subject, given an improvement of the Mn2+ treatment protocols to minimize potential toxic side effects
MULTIMODAL ASSESSMENT OF CETACEAN CENTRAL NERVOUS AUDITORY PATHWAYS WITH EMPHASIS ON FORENSIC DIAGNOSTICS OF ACOUSTIC TRAUMA
Cetaceans encompass some of the world’s most enigmatic species, with one of their greatest adaptations to the marine environment being the ability to “see” by hearing. Their anatomy and behavior are fine-tuned to emit and respond to underwater sounds, which is why anthropogenic noise pollution is likely to affect them negatively. There are many effects of noise on living organisms, and while knowledge on their entire palette and interplay remain incomplete, evidence for insults ranging from acoustic trauma over behavioral changes, to masking and stress, is accumulating. Humans are subject to peak interest in terms of medical research on noise-induced hearing loss. As major health concerns can be expected across species, addressing this problem in free-ranging cetacean populations will lead to a more sustainable management of marine ecosystems, more effective and balanced policies, and successes in conservation. While progress has been made in behavioral monitoring, electrophysiological hearing assessments and post-mortem examination of the inner ear of cetaceans, but very little is known about the neurochemical baseline and neuropathology of their central auditory pathways. In the present work, we reviewed the known effects of sound on cetaceans in both wild and managed settings and explored the value of animal models of neurodegenerative disease. We began by evaluating a row of antibodies associated with neurodegeneration in a more readily available species, the dog, where acute neurological insult could be derived from clinical history. We then set out to systematically validate a key panel of protein biomarkers for the assessment of similar neurodegenerative processes of the cetacean central nervous system. For this, we developed protocols to adequately sample cetacean auditory nuclei, optimized the immunohistochemical workflow, and used Western blot and alignment of protein sequences between the antigen targeted by our antibodies and the dolphin proteome. A Histoscore was used to semi-quantitively categorize immunoreactivity patterns and dolphins by age and presence of pathology. First results indicated significant differences both between sick and healthy, and young and old animals. We then expanded our list of validated antibodies for use in the bottlenose dolphin and the techniques used to assess them in a multimodal, quantitative way. 7T-MRI and stereology were implemented to assess the neuronal, axonal, glial and fiber tract counts in the inferior colliculus and ventral cochlear nucleus of a healthy bottlenose dolphin, which created a baseline understanding of protein expression in these structures, and the influence of tissue processing. This will make a valuable comparison for when positive controls of acoustic trauma would become available. Furthermore, we explored the connectome and neuronal morphology of auditory nuclei and experimented with probe designs and machine learning algorithms to quantify structures of interest. Comparisons with pathological human brains revealed similarities in the configuration of extracellular matrix components to those of a healthy dolphin, in line with existing knowledge on the tolerance to hypoxia in these diving animals. This could have interesting implications in future investigation of the evolutionary development of marine mammal brains, as well as help diversify out-of-the-box approaches to researching human neurodegenerative disease, as is being done with hibernating species. The data and methodologies described herein contribute to the knowledge on neurochemical signature of the cetacean central nervous system. They are intended to facilitate understanding of auditory and non-auditory pathology and build an evidence-based backbone to future policies regarding noise and other form of anthropogenic threats to the marine environment.Cetaceans encompass some of the world’s most enigmatic species, with one of their greatest adaptations to the marine environment being the ability to “see” by hearing. Their anatomy and behavior are fine-tuned to emit and respond to underwater sounds, which is why anthropogenic noise pollution is likely to affect them negatively. There are many effects of noise on living organisms, and while knowledge on their entire palette and interplay remain incomplete, evidence for insults ranging from acoustic trauma over behavioral changes, to masking and stress, is accumulating. Humans are subject to peak interest in terms of medical research on noise-induced hearing loss. As major health concerns can be expected across species, addressing this problem in free-ranging cetacean populations will lead to a more sustainable management of marine ecosystems, more effective and balanced policies, and successes in conservation. While progress has been made in behavioral monitoring, electrophysiological hearing assessments and post-mortem examination of the inner ear of cetaceans, but very little is known about the neurochemical baseline and neuropathology of their central auditory pathways. In the present work, we reviewed the known effects of sound on cetaceans in both wild and managed settings and explored the value of animal models of neurodegenerative disease. We began by evaluating a row of antibodies associated with neurodegeneration in a more readily available species, the dog, where acute neurological insult could be derived from clinical history. We then set out to systematically validate a key panel of protein biomarkers for the assessment of similar neurodegenerative processes of the cetacean central nervous system. For this, we developed protocols to adequately sample cetacean auditory nuclei, optimized the immunohistochemical workflow, and used Western blot and alignment of protein sequences between the antigen targeted by our antibodies and the dolphin proteome. A Histoscore was used to semi-quantitively categorize immunoreactivity patterns and dolphins by age and presence of pathology. First results indicated significant differences both between sick and healthy, and young and old animals. We then expanded our list of validated antibodies for use in the bottlenose dolphin and the techniques used to assess them in a multimodal, quantitative way. 7T-MRI and stereology were implemented to assess the neuronal, axonal, glial and fiber tract counts in the inferior colliculus and ventral cochlear nucleus of a healthy bottlenose dolphin, which created a baseline understanding of protein expression in these structures, and the influence of tissue processing. This will make a valuable comparison for when positive controls of acoustic trauma would become available. Furthermore, we explored the connectome and neuronal morphology of auditory nuclei and experimented with probe designs and machine learning algorithms to quantify structures of interest. Comparisons with pathological human brains revealed similarities in the configuration of extracellular matrix components to those of a healthy dolphin, in line with existing knowledge on the tolerance to hypoxia in these diving animals. This could have interesting implications in future investigation of the evolutionary development of marine mammal brains, as well as help diversify out-of-the-box approaches to researching human neurodegenerative disease, as is being done with hibernating species. The data and methodologies described herein contribute to the knowledge on neurochemical signature of the cetacean central nervous system. They are intended to facilitate understanding of auditory and non-auditory pathology and build an evidence-based backbone to future policies regarding noise and other form of anthropogenic threats to the marine environment
Caracterização das funções neurotrĂłficas da proteĂna precursora de amilĂłide de Alzheimer
Doutoramento em BiomedicinaThe Amyloid Precursor Protein (APP) is a type 1 membrane glycoprotein,
mainly known as the precursor of the amyloid β-peptide, a central player in
Alzheimer’s disease. Nevertheless, APP has been established as a
neuromodulator of developing and mature nervous system. Alterations in the
level or activity of APP and APP fragments seem to play a critical role in
several neurodegenerative and neurodevelopment disorders. APP is a complex
molecule due to the intricate relationships between its intracellular trafficking,
posttranslational modifications, proteolytic cleavages, and multiple protein
interactors. Various studies currently address the physiological roles of APP
and its fragments, but there are contradictory results and missing pieces that
need further work. The main objective of this thesis was to contribute to the
characterization of the role of APP in neuronal differentiation. Particularly, we
focused on mechanisms mediated by APP, its fragment sAPP, and APP
phosphorylation at serine 655.
First, we characterized the APP protein in Retinoic Acid (RA)-induced SHSY5Y
cell differentiation. The comprehensive analysis of this model exposed a
biphasic temporal response: a first early phase (D0-D4), where a sAPP/APP
peak assists the emergence of new processes and their elongation into
neurites; and a second phase (D4-D8) when increased holoAPP protein levels
are necessary to sustain neuritic elongation and stabilization.
In line with our main aim, we subsequently characterized the relationship
between APP and the neurotrophic EGF-EGFR-ERK signaling pathway. We
showed, for the first time, that APP interacts with proEGF, and confirmed the
interaction with EGFR. Furthermore, we showed that combined APP and EGF
have a synergistic effect on neuronal-like differentiation, related to enhanced
ERK1/2 activation, and observed that APP modulates EGFR expression levels
and trafficking. Both ERK1/2 activation and EGFR seem to be modulated by
the APP S655 phosphorylation state, and phosphorylation at this residue
favours dendritogenesis in mice cortical neurons.
Finally, we focused on discovering APP protein interactors dependent on S655
phosphorylation and with a role in neuronal differentiation. SH-SY5Y
differentiated cells, overexpressing APPWt or S655 phosphomutants, were
used to immunoprecipitate the specific APP proteins and their respective
interacting partners, later identified by mass spectrometry. The
dephosphoS655 APP interactome was enriched in functions associated with
cytoskeleton organization, and these cells were particularly associated with
actin remodeling. The phosphoS655 APP interactome included proteins
involved in the regulation of survival and differentiation, and in various signaling
pathways, correlating well with an enhanced neurite outgrowth displayed by
these cells.
We hope that the knowledge here gathered can contribute to a better
comprehension of APP-driven neurotrophic roles and underlying mechanisms.A ProteĂna Precursora de AmilĂłide (APP) Ă© uma proteĂna membranar mais
conhecida por ser precursora do péptido Amilóide β, tendo por isso um papel
central na doença de Alzheimer. Não obstante, a APP tem sido reconhecida
como neuromodulador do sistema nervoso central. Alterações nos nĂveis ou na
atividade da APP e seus fragmentos estão implicadas em diferentes doenças
neurológicas. As relações entre o seu transporte intracelular, modificações
pĂłs-traducionais, corte proteolĂtico, e proteĂnas com as quais interage sĂŁo
complexas e multifacetadas. Talvez por isso, estudos focados no papel
fisiológico da APP apresentem resultados contraditórios e muitas questões em
aberto. O objetivo deste trabalho consistiu na caracterização do papel
fisiológico da APP na diferenciação neuronal. Particularmente, focámo-nos nos
mecanismos mediados pela APP e fragmento sAPP, e a fosforilação da APP
no resĂduo serina 655.
Inicialmente, caracterizámos a proteĂna APP ao longo da diferenciação de
células SH-SY5Y com ácido retinóico (RA). A análise sistemática deste modelo
permitiu delimitar uma resposta bifásica: na primeira fase (D0-D4), um pico de
sAPP/APP acompanha o aparecimento de novos processos e o crescimento a
neurites; na segunda fase (D4-D8) o aumento nos nĂveis da APP suporta o
crescimento e manutenção das neurites.
Caracterizámos posteriormente a relação entre a APP e a via de sinalização
EGF-EGFR-ERK na diferenciação neuronal. Demonstrámos, pela primeira vez,
que a APP interage com o proEGF, e confirmámos a sua ligação ao EGFR.
Adicionalmente, observámos que a APP e o EGF têm um efeito sinérgico na
diferenciação tipo-neuronal e aumento da ativação da ERK1/2, e que a APP
afeta os nĂveis e transporte do EGFR. Estes mecanismos sĂŁo modulados pela
fosforilação da APP na S655, que favorece a dendritogénese em neurónios
corticais de ratinho.
Por Ăşltimo, focámo-nos na identificação de proteĂnas interatoras da APP
dependentes da fosforilação em S655 e com função na diferenciação neuronal.
Usando células SH-SY5Y diferenciadas e a sobrexpressar a APPWt ou
fosfomutantes da S655, imunoprecipitámos as diferentes APPs e seus
interatores, posteriormente identificados por espectrometria de massa. O
interatoma da APP desfosforilada Ă© enriquecido em funções associadas Ă
organização do citoesqueleto, levando a uma maior reorganização da actina. O
interatoma da APP fosforilada incluĂ proteĂnas envolvidas na regulação de
sobrevivência e diferenciação, e em várias vias de sinalização, o que se
correlaciona com o favorecimento de neurites nestas células.
Com este trabalho esperamos ter contribuĂdo para uma melhor compreensĂŁo
do papel neurotrĂłfico da APP e dos mecanismos subjacentes a este