Stroke is cerebrovascular injury that has been reported to be the third leading cause of death and the first leading cause of disability in the world (W. H.O. 2007). Currently, there is only one FDA approved treatment for stroke which is recombinant tissue plasminogen activator. This treatment has a narrow therapeutic window of three hours after ischemic stroke and can adversely cause the production of oxygen free radicals and intracranial hemorrhage. These limitations result in only 2-3% of all stroke victims as being candidates for this therapy as many patients do not arrive at the hospital in time to receive treatment, are not properly diagnosed, or do not know that they have had a stroke within this three hour time period. The purpose of these experiments was to elucidate alternative targets of stroke for the benefit of developing new treatments that stimulate neuroprotective and anti-inflammatory effects at the site of injury. It has been shown that transfusion of human umbilical cord blood cells up to 48 hours after stroke significantly reduces infarction and we have examined other targets that mimic these effects. We have shown that sigma receptor activation by DTG, a high affinity universal sigma agonist, reduces infarct volume when administered 24 hours after stroke. This suggests that modulation of neurodegenerative and inflammatory responses can extend the therapeutic window of treatment. For the first time, evidence is provided that shows that the spleen enhances the neurodegeneration caused by stroke as splenectomy prior to stroke profoundly decreased infarction volume. Finally, we studied signaling mechanisms of the splenic reaction to stroke and determined that this response is not directly dependent on neurotransmission via the splenic nerve. Denervation of the spleen prior to stroke showed no changes in neurodegenerative load at the site of injury in rat brains when compared to those subjected to stroke only. Overall, these experiments provide evidence showing that targets mediating neuroprotective and anti-inflammatory effects can lead to novel therapeutic interventions of stroke.

Fetal alcohol spectrum disorders (FASD) are caused by alcohol exposure during pregnancy and is the leading cause of mental retardation. Alcohol exposure during development results in the loss of neurons in the developing brain. The underlying molecular mechanisms are unclear and there currently is no cure for FASD. Ethanol-induced neuronal death is accompanied by neuroinflammation. Chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) are critical mediators of neuroinflammation and microglial activation. Using a third trimester equivalent mouse model of ethanol exposure, we found that treatment of Bindarit (MCP-1 synthesis inhibitor) and RS504393 (CCR2 antagonist) significantly reduced ethanol-induced microglia activation/neuroinflammation, and neuroapoptosis in the developing brain. Moreover, ethanol plus MCP-1 caused more neuronal death in a neuron/microglia co-culture system than neuronal culture alone, and Bindarit and RS504393 attenuated ethanol-induced neuronal death in the co-culture system. Ethanol activated TLR4 and GSK3β, two key mediators of microglial activation in the brain and cultured microglial cells (SIM-A9). Blocking MCP-1/CCR2 signaling attenuated ethanol-induced activation of TLR4 and GSK3β. Further, we determined whether knocking out of MCP-1/CCR2 ameliorates neonatal alcohol exposure-induced long-lasting behavioral deficits in adolescent and adult mice. C57BL/6 and MCP-1-/-/CCR2-/- mice were exposed to alcohol (5 g/kg) by subcutaneously injection on PD4. A series of behavioral tests including Open Field (PD 35-36 and PD 70-71), Rotor-Rod (PD 38 and PD 73), Balance Beam (PD 40 and PD75) and Morris Water Maze (PD 42 and PD77) were performed in the adolescence and adulthood. We found that MCP-1-/-/CCR2-/- mice were resistant to neonatal alcohol exposure-induced deficits in motor function in the Rotor-Rod and Balance Beam tests; MCP-1 and CCR2 deficiency also protected mice against neonatal ethanol exposure induced long lasting deficits in learning and memory in the Morris Water Maze testing. Collectively, these results suggest that MCP-1/CCR2 signaling plays an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain and also plays an important role in developmental alcohol exposure induced long-lasting behavioral deficits in adolescence and adulthood.

SARM1 is an injury-induced nicotinamide adenine dinucleotide nucleosidase (NADase) that was previously shown to promote axonal degeneration in response to traumatic, toxic, and excitotoxic stressors. This raises the question of whether a SARM1-dependent program of axonal degeneration is central to a common pathway contributing to disease burden in neurological disorders. The degree to and mechanism by which SARM1 inactivation decreases the pathophysiology of such disorders is of interest to establish the rationale to pursue SARM1 as a therapeutic target. In this study, we compare the course and pathology of experimental autoimmune encephalomyelitis (EAE) in Sarm1-knockout (KO) mice and wild-type littermates to test the contribution of SARM1-dependent axonal degeneration specifically in the context of chronic, immune-mediated central nervous system (CNS) inflammation. The question of whether SARM1 loss in Sarm1-KO mice would inhibit, promote, or have a negligible impact on EAE-induced axonal degeneration and more broadly CNS inflammation was explored using a variety of analyses: quantification of clinical score in a chronic EAE model, CNS immune infiltrate profile, axon initial segment morphology in layer V cortical neurons, axonal transport disruption and transection in the lumbar spinal cord. Additionally, we have proposed a method for detecting SARM1 activation in situusing a novel SARM1-mCitrine bimolecular fluorescence complementation (BiFC) technique. Successful implementation of such a molecular tool would allow for a detailed, mechanistic approach to enhance our understanding of upstream intracellular signals that trigger SARM1 activation.

Abstract Undefined severe neurological and multi-organ diseases are rare as single diseases, but as a group of diseases, they are responsible for significant morbidity, impaired quality of life and mortality, emphasizing the importance of neuroscience research and its translation into novel diagnostic and treatment strategies. Molecular karyotyping and whole-exome sequencing were used to identify three novel disease-causing genes, GLE1, NHLRC2 and MYH7B, in Northem Finnish families having children with undefined progressive neuromuscular diseases. Functional studies on GLE1, NHLRC2, and MYH7B were conducted in order to understand better the impact of these mutations. The studies revealed that the cellular localization of GLE1 was impaired due to a mutation in the coding gene. The NHLRC2 is involved in many biological processes and its dysfunction has a role in the development of a novel FINCA disease and in fibrosis. Furthermore, mutations in MYH7B in the myosin family have now been connected to encephalomyopathies. Mutations in GLE1, NHLRC2 and MYH7B are involved in encephalomyopathies and neurodegeneration, stressing the important role of these genes in normal psychomotor development Analyses of these previously uncharacterized disease-causing gene mutations provided new insights into the etiologies behind these diseases, representing a relevant starting point for resolving the pathomechanisms underpinning these disorders. The newly-discovered human disease-causing genes and the novel phenotypes of childhood onset neuromuscular diseases provide the possibility for offering the relevant families preclinical diagnostics and may be beneficial in the identification of similar clinical phenotypes all around the world. / Tiivistelmä Yksittäiset, määrittelemättömät, vaikeat neurologiset monielinsairaudet ovat harvinaisia. Sen sijaan neurologisten ja monielinsairauksien alle ryhmittyvät taudit ovat merkittävä syy useisiin sairauksiin, jotka heikentävät elämänlaatua ja aiheuttavat kuolleisuutta. Tästä johtuen neurotieteiden tutkimus ja saatujen tulosten soveltaminen diagnostiikassa ja hoitomuotojen kehittämisessä on hyvin tärkeää. Molekyylikaryotyypitys- ja eksomisekvensointi-menetelmiä hyödynnettiin etsittäessä taudin syytä eteneville neuromuskulaarisairauksille pohjoissuomalaisissa perheissä. Tutkimuksessa tehtiin lisäksi funktionaalisia kokeita GLE1-, NHLRC2- ja MYH7B-proteiineilla, jotta ymmärrettäisiin paremmin löydettyjen mutaatioiden vaikutus potilaiden sairauksiin. Havaittiin, että GLE1-mutaatio vaikutti proteiinin solunsisäiseen paikantumiseen. NHLRC2-proteiini puolestaan on mukana useissa solun biologisissa prosesseissa ja sen toiminnanhäiriö vaikuttaa FINCA-taudin ja fibroosin kehittymiseen. MYH7B-myosiinigeenimutaatio puolestaan yhdistettiin ensimmäistä kertaa enkefalomyopatiaan. Havaittujen tautigeenien; GLE1, NHLRC2 ja MYH7B, vaikutus enkefalomyopatioissa ja neurodegeneraatiossa kertoo, että kyseisillä geeneillä on hyvin todennäköisesti tärkeä rooli ihmisen kehityksessä. Kyseisten, aiemmin tuntemattomien sairautta-aiheuttavien geenimutaatioiden analysointi lisäsi tietoa sairauksien etiologiasta ja loi pohjan tautimekanismien ratkaisemiselle tulevaisuudessa. Työssä esitettyjä uusia sairautta-aiheuttavia geenejä ja uusia karakterisoituja lapsuusiän neuromuskulaarisairauksien ilmiasuja voidaan hyödyntää perheille tarjotun sikiödiagnostiikan lisäksi myös muiden potilaiden samankaltaisen taudinkuvan diagnosoinnissa maailmanlaajuisesti.

<p>Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive disorder characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord. The patients usually die within 3-5 years after onset. The full etiology of ALS is unknown and many hypotheses have been proposed to explain the neurodegeneration. However, basic mechanisms of cellular function such as transmethylation and polyamine metabolism have not been extensively studied in ALS. Transmethylation reactions are very important in the synthesis of substrates such as proteins, neurotransmitters, DNA and RNA. The polyamines, putrescine, spermidine and spermine, are involved in essential functions such as cellular growth, proliferation and differentiation.</p><p>An initial study in this thesis concerned the process of neuronal death (apoptosis) in ALS spinal cord. The results showed increased levels of an apoptosis-stimulating protein and increased levels of DNA fragmentation indicative of an apoptotic process in the tissue. A comparative study of MAT-enzyme activity in spinal cord from different mammalian species was undertaken to provide a background for future studies on transmethylation and neurodegeneration. Transmethylation reactions were found altered in erythrocytes from males with ALS but not in spinal cord from ALS patients as compared to controls. An adaptation of previously described polyamine assays was made for the study of polyamines in ALS spinal cord. The method was validated and applied for polyamine analysis in human materials of different characteristics. Determination of polyamines in control and ALS spinal cords showed no major differences. However, in female ALS patients, significantly increased spermidine and spermine levels were observed in ventral horn regions. These gender-related alterations in transmethylation and polyamine metabolism are of interest since there is a male preponderance for the disease.</p><p>The lack of major differences in polyamine levels between ALS and control spinal cord suggests a maintained regulation of polyamines at the end stage of this neurodegenerative disease.</p>

<p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.</p>

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of unesterified cholesterol in late endosomes/lysosomes (LE/Ls), leading to progressive neurodegeneration and premature death. Microglia are resident immune cells of the central nervous system, which upon activation can secrete potentially neurotoxic molecules such as tumor necrosis factor-alpha (TNFα). Inappropriate activation of microglia has been implicated in NPC disease. Primary microglia cultures from the cerebral cortex of Npc1-/- mice have an altered cholesterol distribution characteristic of NPC-deficient cells. Immunocytochemical analysis revealed increased TNFα staining in Npc1-/- microglia. However, Npc1-/- and Npc1+/+ microglia showed similar mRNA levels of pro-inflammatory cytokines and similar levels of TNFα secretion. To determine whether Npc1-/- microglia contribute to neuron death in NPC disease, microglia were co-cultured with cerebellar granule cells. Surprisingly, the extent of neuronal death was the same in neurons cultured with Npc1+/+ or Npc1-/- microglia. Thus, Npc1-/- microglia have an altered phenotype compared to Npc1+/+ microglia, but this does not lead to neuron death in an in vitro co-culture system. Treatment options for NPC disease remain limited. A consequence of cholesterol sequestration in the LE/Ls, is that cholesterol movement to the endoplasmic reticulum, where cholesterol metabolism is regulated, is impaired. Cyclodextrin (CD), a compound that binds cholesterol, has recently been found to delay the onset of neurological symptoms and prolong life of Npc1-/- mice. Since the brain consists of both neurons and glia, it remains unclear if CD acts directly on neurons and/or other cells in the brain. Neurons cultured from the cerebellum and astrocytes cultured from the cortex of Npc1-/- mice were treated with a low dose (0.1mM) of CD. This treatment decreased cholesterol sequestration and decreased the rate of cholesterol synthesis in Npc1-/- neurons and astrocytes. CD also decreased mRNAs encoding proteins involved in cholesterol synthesis in Npc1-/- neurons and increased genes involved in cholesterol efflux in Npc1-/- astrocytes. Moreover, CD increased cholesterol esterification in Npc1-/- astrocytes. These results suggest that cholesterol trapped in LE/Ls in Npc1-/- neurons and astrocytes was released by CD treatment and reached the ER, thereby regulating cholesterol homeostasis. / Experimental Medicine

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive disorder characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord. The patients usually die within 3-5 years after onset. The full etiology of ALS is unknown and many hypotheses have been proposed to explain the neurodegeneration. However, basic mechanisms of cellular function such as transmethylation and polyamine metabolism have not been extensively studied in ALS. Transmethylation reactions are very important in the synthesis of substrates such as proteins, neurotransmitters, DNA and RNA. The polyamines, putrescine, spermidine and spermine, are involved in essential functions such as cellular growth, proliferation and differentiation. An initial study in this thesis concerned the process of neuronal death (apoptosis) in ALS spinal cord. The results showed increased levels of an apoptosis-stimulating protein and increased levels of DNA fragmentation indicative of an apoptotic process in the tissue. A comparative study of MAT-enzyme activity in spinal cord from different mammalian species was undertaken to provide a background for future studies on transmethylation and neurodegeneration. Transmethylation reactions were found altered in erythrocytes from males with ALS but not in spinal cord from ALS patients as compared to controls. An adaptation of previously described polyamine assays was made for the study of polyamines in ALS spinal cord. The method was validated and applied for polyamine analysis in human materials of different characteristics. Determination of polyamines in control and ALS spinal cords showed no major differences. However, in female ALS patients, significantly increased spermidine and spermine levels were observed in ventral horn regions. These gender-related alterations in transmethylation and polyamine metabolism are of interest since there is a male preponderance for the disease. The lack of major differences in polyamine levels between ALS and control spinal cord suggests a maintained regulation of polyamines at the end stage of this neurodegenerative disease.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.

Patients suffering from different forms of neurodegenerative diseases, such as: Creutzfeldt Jacob Disease (CJD), Alzheimer disease (AD), mild cognitive impairment (MCI), frontotemporal dementia and Parkinson’s disease (PD) were examined with Positron Emission Tomography (PET) and the combination of different radiotracers: 15O-water, N-[11C-methyl]-L-deuterodeprenyl (DED), [18F] 2-fluorodeoxyglucose: (FDG), N-methyl-[11C]2-(4-methylaminophenyl)-6-hydroxybenzothiazole (PIB) and L-[11C]-3,4-dihydroxiphenyl-alanine (DOPA). The radiotracers and the combinations of different radiotracers were selected with the intention to detect, in the brain, patterns of neuronal dysfunction, astrocytosis, axon degeneration or protein aggregation (amyloid), in the brain which are pathognomonic for specific diseases and may contribute to improve clinical differential diagnoses. Examinations in healthy volunteers were performed to allow comparisons with patients. In addition, animal studies were conducted to complement the information. In some cases, the PET findings could be compared with the results of autopsies. In contrast to the micropathology, in which only a limited part of a tissue (obtained post-mortem or by biopsy) is inspected, one PET acquisition provides an image of the whole system (e.g.: the brain and the cerebellum). This form of imaging pathology is “in vivo”, where the examination is innocuous for the patient. This thesis is an attempt to stimulate the development of new tracers, new tracer combinations and methods that directly or indirectly describe the anatomo-physiopathological changes produced in the brain in neurodegenerative diseases. A better description of different diseases can be obtained, confirming or questioning the clinical diagnoses and widening our understanding of the mechanisms underlying neurodegeneration. Different pathologies can produce similar symptoms and thus causing confusion regarding clinical diagnosis. The used PET combinations improved the accuracy of the diagnoses. The incipient knowledge emerging from a new neuroimaging pathology in combination with other disciplines may open the way to new classifications of dementias and neurodegenerative diseases based on an “in vivo” pathology.