This study investigated apoptosis in a transgenic mouse model of ALS. The researchers found:
1) DNA fragmentation, a marker of apoptosis, was significantly higher in the brain regions of mice expressing a mutant SOD1 gene compared to control mice, as shown by TUNEL staining and ELISA assays.
2) Double staining showed many TUNEL-positive motoneurons and glial cells in the spinal cord and brainstem of mutant SOD1 mice, but not controls.
3) Electron microscopy confirmed neuronal and glial apoptosis in these regions through characteristic morphological features in mutant SOD1 mice.
4) TUNEL-positive neurons were also observed in the motor and sensory cortices of mutant
1) Researchers screened 40,000 compounds and identified TRO19622 as a potential drug candidate for treating amyotrophic lateral sclerosis (ALS).
2) In vitro, TRO19622 promoted motor neuron survival in a dose-dependent manner and rescued motor neurons from death.
3) In animal models of ALS and nerve damage, TRO19622 improved motor performance, delayed disease onset, extended survival, and promoted nerve regeneration.
This document summarizes Hannah Shapero's research project on the effects of a sut-2 mutation in Caenorhabditis elegans worms expressing human TDP-43. The research found that a point mutation in the sut-2 gene led to a partial amelioration of the uncoordinated phenotype seen in worms expressing human TDP-43 alone. This suggests the sut-2 mutation can suppress some of the toxic effects caused by TDP-43 protein aggregates, which are implicated in amyotrophic lateral sclerosis and other neurodegenerative diseases.
Endothelial Cell Mediated Delay of Blood Brain Barrier Recovery Following Tra...Arthur Stem
TBI is the leading cause of death among young adults and children in the developed world, accounting for over 50,000 deaths per year. [12] TBI results in a sleuth of poor health outcomes, including hemorrhaging, seizures, neural edema, neural inflammation, and cognitive and emotional disabilities. All of these outcomes are a direct result of fundamental degradation of the BBB over a time course post TBI. [1] [12] The BBB is an integral structure that forms around the microvascular of the cerebral cavity. Endothelial cells form the basal membrane through which strictly controlled movement of molecules is observed between the extravascular and intravascular space across this basal membrane. This basal membrane is maintained by endothelial cells, having tight junctions between them to make up the pores through which transport of molecules can occur between the brain and microvasculature. These tight junctions are maintained through cross-talk between the endothelial cells and supporting neurons such as astrocytes and pericytes. [2] A multitude of proteins make up the tight junctions between the endothelial cells, including six main scaffolding structures Claudins 1, 3, and 5, ZO-1, Occludins, and Cadherins. [3] VEGF release following trauma induces endothelial cells to release matrix metalloproteinases (MMPs), in particular MMP9, which can catalyze the N-terminal amino acids that compose the tight junction protein ZO-1. [10] [11] MMP9 when in circulation is also known to activate tumor necrosis factor alpha (TNFɑ) which in turn upregulates transcription of MMP9, creating a positive feedback loop. [11] The management of MMP production is three fold, transcription, proenzyme activation, and substrate inhibition. [11] In our study, it is proenzyme activation via TP that is the focus and how that affects the overall transcription levels of the tight junction proteins within the endothelial cells and astrocytes.
10. jULIETA gONZALEZ. Xerostomia no solo un problema de aguacrea-autoinmunidad
The document summarizes research on dry mouth (xerostomia) in Sjögren's syndrome patients. It discusses two models - the apoptotic model where immune system alterations destroy salivary glands, and the non-apoptotic model where reduced water transport causes low saliva flow. Studies found altered expression of genes and mucins involved in saliva production and composition in patients. Inflammatory cytokines may impact mucin sulfation levels correlated with dryness symptoms. The research suggests dryness in Sjögren's syndrome involves more than gland destruction and may be linked to changes in mucin production and composition affecting water transport.
Modelling gender-specific regulation of tau in Alzheimer’s diseaseEnrico Glaab
Public transcriptomic studies have shown that several genes display pronounced gender differences in their expression in the human brain, which may influence the manifestations and risk for neuronal disorders. Here, we apply a transcriptome-wide analysis to discover genes with gender-specific expression and significant alterations in public postmortem brain tissue from Alzheimer’s disease (AD) patients compared to controls. We identify the sex-linked ubiquitin-specific peptidase 9 (USP9) as an outstanding candidate gene with highly significant expression differences between the genders and male-specific underexpression in AD. Since previous studies have shown that USP9 can modulate the phosphorylation of the AD-associated protein MAPT, we investigate functional associations between USP9 and MAPT in further detail. After observing a high positive correlation between the expression of USP9 and MAPT in the public transcriptomics data, we show that USP9 knockdown results in significantly decreased MAPT expression in a DU145 cell culture model and a concentration-dependent decrease for the MAPT orthologs mapta and maptb in a zebrafish model. From the analysis of microarray and qRT-PCR experiments for the knockdown in DU145 cells and prior knowledge from the literature, we derive a data-congruent model for a USP9-dependent regulatory mechanism modulating MAPT expression via BACH1 and SMAD4. Overall, the analyses suggest USP9 may contribute to molecular gender differences observed in tauopathies and provide a new target for intervention strategies to modulate MAPT expression.
See associated publication: http://link.springer.com/article/10.1007%2Fs12035-016-0299-z
This document summarizes a study investigating the role of intracellular calcium levels in alcohol-induced death of cerebellar granule neurons (CGN). The study found that alcohol exposure caused a rapid, dose-dependent increase in intracellular calcium levels in CGN that persisted over time. Inhibiting the rise in intracellular calcium, either by blocking calcium release from intracellular stores or by chelating calcium, prevented both the alcohol-induced increase in intracellular calcium and neuronal death. The sustained rise in intracellular calcium may be a key factor in the mechanism of alcohol-induced neuronal death.
This study examined the effects of perforin, a pore-forming protein, on oligodendrocytes cultured from SJL mice. Oligodendrocytes were exposed to perforin for up to 2.5 hours and examined using microscopy. The results showed that the majority of oligodendrocytes were killed within 60-90 minutes via pore expansion and membrane disruption. Structural features included cell body swelling, fenestration and fragmentation of membranes and processes, cytoplasmic vacuolation, and breakdown of the nuclear envelope. These patterns of damage resembled those seen in multiple sclerosis lesions. The findings suggest that perforin may play an important role in demyelination in multiple sclerosis.
Presentation made by Abeliovich and Rhinn on the 20th of April, 2017, at the live webinar hosted by Alzforum: http://www.alzforum.org/webinars/webinar-cortex-aging-too-fast-blame-tmem106b-and-progranulin
1) Researchers screened 40,000 compounds and identified TRO19622 as a potential drug candidate for treating amyotrophic lateral sclerosis (ALS).
2) In vitro, TRO19622 promoted motor neuron survival in a dose-dependent manner and rescued motor neurons from death.
3) In animal models of ALS and nerve damage, TRO19622 improved motor performance, delayed disease onset, extended survival, and promoted nerve regeneration.
This document summarizes Hannah Shapero's research project on the effects of a sut-2 mutation in Caenorhabditis elegans worms expressing human TDP-43. The research found that a point mutation in the sut-2 gene led to a partial amelioration of the uncoordinated phenotype seen in worms expressing human TDP-43 alone. This suggests the sut-2 mutation can suppress some of the toxic effects caused by TDP-43 protein aggregates, which are implicated in amyotrophic lateral sclerosis and other neurodegenerative diseases.
Endothelial Cell Mediated Delay of Blood Brain Barrier Recovery Following Tra...Arthur Stem
TBI is the leading cause of death among young adults and children in the developed world, accounting for over 50,000 deaths per year. [12] TBI results in a sleuth of poor health outcomes, including hemorrhaging, seizures, neural edema, neural inflammation, and cognitive and emotional disabilities. All of these outcomes are a direct result of fundamental degradation of the BBB over a time course post TBI. [1] [12] The BBB is an integral structure that forms around the microvascular of the cerebral cavity. Endothelial cells form the basal membrane through which strictly controlled movement of molecules is observed between the extravascular and intravascular space across this basal membrane. This basal membrane is maintained by endothelial cells, having tight junctions between them to make up the pores through which transport of molecules can occur between the brain and microvasculature. These tight junctions are maintained through cross-talk between the endothelial cells and supporting neurons such as astrocytes and pericytes. [2] A multitude of proteins make up the tight junctions between the endothelial cells, including six main scaffolding structures Claudins 1, 3, and 5, ZO-1, Occludins, and Cadherins. [3] VEGF release following trauma induces endothelial cells to release matrix metalloproteinases (MMPs), in particular MMP9, which can catalyze the N-terminal amino acids that compose the tight junction protein ZO-1. [10] [11] MMP9 when in circulation is also known to activate tumor necrosis factor alpha (TNFɑ) which in turn upregulates transcription of MMP9, creating a positive feedback loop. [11] The management of MMP production is three fold, transcription, proenzyme activation, and substrate inhibition. [11] In our study, it is proenzyme activation via TP that is the focus and how that affects the overall transcription levels of the tight junction proteins within the endothelial cells and astrocytes.
10. jULIETA gONZALEZ. Xerostomia no solo un problema de aguacrea-autoinmunidad
The document summarizes research on dry mouth (xerostomia) in Sjögren's syndrome patients. It discusses two models - the apoptotic model where immune system alterations destroy salivary glands, and the non-apoptotic model where reduced water transport causes low saliva flow. Studies found altered expression of genes and mucins involved in saliva production and composition in patients. Inflammatory cytokines may impact mucin sulfation levels correlated with dryness symptoms. The research suggests dryness in Sjögren's syndrome involves more than gland destruction and may be linked to changes in mucin production and composition affecting water transport.
Modelling gender-specific regulation of tau in Alzheimer’s diseaseEnrico Glaab
Public transcriptomic studies have shown that several genes display pronounced gender differences in their expression in the human brain, which may influence the manifestations and risk for neuronal disorders. Here, we apply a transcriptome-wide analysis to discover genes with gender-specific expression and significant alterations in public postmortem brain tissue from Alzheimer’s disease (AD) patients compared to controls. We identify the sex-linked ubiquitin-specific peptidase 9 (USP9) as an outstanding candidate gene with highly significant expression differences between the genders and male-specific underexpression in AD. Since previous studies have shown that USP9 can modulate the phosphorylation of the AD-associated protein MAPT, we investigate functional associations between USP9 and MAPT in further detail. After observing a high positive correlation between the expression of USP9 and MAPT in the public transcriptomics data, we show that USP9 knockdown results in significantly decreased MAPT expression in a DU145 cell culture model and a concentration-dependent decrease for the MAPT orthologs mapta and maptb in a zebrafish model. From the analysis of microarray and qRT-PCR experiments for the knockdown in DU145 cells and prior knowledge from the literature, we derive a data-congruent model for a USP9-dependent regulatory mechanism modulating MAPT expression via BACH1 and SMAD4. Overall, the analyses suggest USP9 may contribute to molecular gender differences observed in tauopathies and provide a new target for intervention strategies to modulate MAPT expression.
See associated publication: http://link.springer.com/article/10.1007%2Fs12035-016-0299-z
This document summarizes a study investigating the role of intracellular calcium levels in alcohol-induced death of cerebellar granule neurons (CGN). The study found that alcohol exposure caused a rapid, dose-dependent increase in intracellular calcium levels in CGN that persisted over time. Inhibiting the rise in intracellular calcium, either by blocking calcium release from intracellular stores or by chelating calcium, prevented both the alcohol-induced increase in intracellular calcium and neuronal death. The sustained rise in intracellular calcium may be a key factor in the mechanism of alcohol-induced neuronal death.
This study examined the effects of perforin, a pore-forming protein, on oligodendrocytes cultured from SJL mice. Oligodendrocytes were exposed to perforin for up to 2.5 hours and examined using microscopy. The results showed that the majority of oligodendrocytes were killed within 60-90 minutes via pore expansion and membrane disruption. Structural features included cell body swelling, fenestration and fragmentation of membranes and processes, cytoplasmic vacuolation, and breakdown of the nuclear envelope. These patterns of damage resembled those seen in multiple sclerosis lesions. The findings suggest that perforin may play an important role in demyelination in multiple sclerosis.
Presentation made by Abeliovich and Rhinn on the 20th of April, 2017, at the live webinar hosted by Alzforum: http://www.alzforum.org/webinars/webinar-cortex-aging-too-fast-blame-tmem106b-and-progranulin
This document summarizes a project investigating whether mitochondrial DNA-encoded oxidative phosphorylation (OXPHOS) transcripts are dysregulated in the blood of patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared to controls. The student researcher designed primers, conducted quantitative real-time PCR on blood samples from controls and patients, and found several mtDNA-encoded OXPHOS transcripts were significantly more abundant in MCI and AD patients. This suggests peripheral changes in mitochondrial gene expression occur early in AD and could provide biomarkers for early diagnosis and monitoring disease progression and treatment responses.
Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
This document summarizes a study on developing a novel protein interaction platform to study neurodegeneration. The study cultured neuronal stem cells to form neurospheres, which were used as a model system. Lentiviral transfection was optimized to introduce target genes stably into the neurospheres. The goal was to analyze protein interactions of Alzheimer's disease proteins by co-immunoprecipitation of neurosphere cultures expressing tagged target proteins. This model aims to further the understanding of molecular mechanisms in neurodegenerative disorders like Alzheimer's disease.
1) The study characterized host vascularization of transplanted human neural stem cell grafts in porcine spinal cords. Twelve grafts of human neural progenitor cells were transplanted into three pigs' spinal cords.
2) Six weeks after transplantation, the pigs were sacrificed and the grafts were analyzed. Graft survival ranged from 0-65% and inversely correlated with host microvascular density in the grafts. More vascularized grafts had less cell survival.
3) Vessels in the grafts expressed vascular endothelial growth factor and contained proliferating endothelial cells and immune cells, which may impact graft survival. The findings could influence future immunosuppression regimens for cell transplantation.
Genetic Insights Into Multiple Sclerosis PathogenesisAaron Sparshott
A segment of a group presentation reflecting upon some of the genetic components that may contribute to Multiple Sclerosis pathogenesis.
IL2Rα and IL7Rα were the two genes of focus.
(This presentation was originally done for Semester 2 , 2008)
20140824 Abnormalities in human pluripotent cells due to reprogramming mechan...Alim Polat
This study compares genetic and epigenetic abnormalities in three types of human pluripotent stem cells: induced pluripotent stem cells (iPS cells), nuclear transfer embryonic stem cells (NT ES cells), and in vitro fertilization embryonic stem cells (IVF ES cells). The researchers found that both iPS cells and NT ES cells derived from the same somatic cells contained similar numbers of copy number variations, whereas DNA methylation and gene expression profiles of NT ES cells more closely matched those of IVF ES cells. The results suggest that human somatic cells can be faithfully reprogrammed to pluripotency by somatic cell nuclear transfer and may be better suited than iPS cells for cell replacement therapies due to avoiding
This research article describes the generation of new mouse models of partial trisomy and monosomy of human chromosome 21. The models contain extra or missing copies of a 9.4 Mb region on mouse chromosome 16 that is syntenic to a region on human chromosome 21. Mice with an extra copy (trisomy model) showed impaired locomotion and increased muscle strength/mass, while mice missing a copy (monosomy model) showed the opposite phenotype. Gene expression analysis revealed that genes related to muscle energy metabolism and mitochondria were downregulated in the trisomy mice and upregulated in the monosomy mice. This correlated with changes in mitochondrial proliferation and function in skeletal muscle, providing insight into the muscle and locom
1. The study investigated whether an axonal targeting sequence exists in the survival motor neuron (SMN) protein by analyzing a truncated form of SMN (SMN236) containing exons 2, 3 and 6.
2. The results showed that SMN236 co-localized with Gemin3 in axonal granules in neuroblastoma cells, similarly to full-length SMN. Granules containing SMN236 also exhibited bidirectional movement along axons comparable to granules containing full-length SMN.
3. These findings suggest that an axonal targeting sequence is located within exons 2, 3 and/or 6 of SMN. Identifying this sequence could help further characterize SMN's role
CODAS syndrome is a rare multi-system developmental disorder characterized by cerebral, ocular, dental, auricular, and skeletal anomalies. The researchers identified four mutations in the LONP1 gene in ten individuals with CODAS syndrome from three ancestral backgrounds. LONP1 encodes the mitochondrial Lon protease, which is involved in protein quality control and respiratory complex assembly in mitochondria. The mutations cluster in the AAA+ domain near the ATP-binding pocket and result in defects in ATP-dependent proteolysis. Lymphoblastoid cell lines from affected individuals show swollen mitochondria, aggregated cytochrome c oxidase subunit II, and reduced mitochondrial function, linking LONP1 mutations to CODAS syndrome.
This document discusses oligodendrocytes, the cells that form myelin in the central nervous system. It describes their role in development, diseases like multiple sclerosis where remyelination fails, and potential therapies to promote remyelination. A clinical trial was conducted using neural progenitor cell transplantation to treat Pelizaeus-Merzbacher disease, a fatal leukodystrophy caused by myelin deficiency. Research suggests the Wnt signaling pathway inhibits oligodendrocyte differentiation and myelination, and a small molecule Wnt inhibitor was found to accelerate remyelination in mice.
This study identified FUS, a gene frequently mutated in ALS, as autoregulating its own expression through alternative splicing of its pre-mRNA. The study found that FUS binds to and represses the splicing of its own exon 7. This causes the production of an exon 7-skipped splice variant that triggers nonsense-mediated decay, reducing FUS protein levels. Expression of ALS-linked FUS mutants compromised this autoregulation by reducing exon 7 repression and nuclear localization of FUS. This suggests deficient FUS autoregulation could exacerbate cytoplasmic accumulation of FUS in ALS through a feed-forward loop mechanism.
This article describes research identifying small molecule pan-caspase inhibitors that could potentially treat Huntington's disease. The researchers screened compounds against caspase-3 and -6 to identify three novel inhibitors that blocked huntingtin protein cleavage by these caspases. Testing in Huntington's disease models found that the irreversible inhibitors suppressed toxicity from mutant huntingtin protein and rescued neurons from cell death. The results support that caspase-mediated cleavage of huntingtin plays a critical role in Huntington's disease pathogenesis, and inhibiting caspases may be a promising therapeutic strategy.
This document reports on a study that tested the effects of isoxazole 9 (Isx-9), a small synthetic molecule, on adult hippocampal neurogenesis in rats. The study found that administering Isx-9 for 14 days potentiated cell proliferation and increased the number of immature neurons in the hippocampal dentate gyrus. Isx-9 treatment also completely reversed the reduction in cell proliferation and neuronal commitment observed in vehicle-treated animals that were subjected to repeated handling and injections. These findings demonstrate that Isx-9 has promising pro-neurogenic properties and could help mitigate stress-induced deficits in adult hippocampal neurogenesis.
Mitochondrial diseases are caused by defects in mitochondrial structure or enzymes that result in deficient energy production. They can affect multiple organ systems and occur across all age groups. Mitochondrial DNA mutations can be inherited from the mother and nuclear DNA mutations can affect mitochondrial proteins or DNA maintenance. Common mitochondrial diseases include MELAS, MERRF, and Leigh syndrome. Mitochondrial dysfunction has also been implicated in aging and common diseases like heart disease and Parkinson's.
This study used an AAV vector to express clarin-1 (CLRN1) in the mouse retina to determine its subcellular localization. CLRN1 was expressed in all major retinal cell types when driven by a ubiquitous promoter. In photoreceptors, CLRN1 localized mainly to the inner segment and outer plexiform layer, similar to other usher proteins. High-titer subretinal delivery led to loss of retinal function, suggesting a critical limit for CLRN1 expression. The results imply CLRN1 expression may be supported by multiple retinal cells and that the dose, promoter, and delivery method need optimization for USH3 gene therapy.
1) Mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 (NL2) were found to exhibit increased anxiety-like behavior.
2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
This study aimed to determine if mutations in the human cystatin M/E gene (CST6) contribute to harlequin ichthyosis (HI) by sequencing the entire coding region and intron-exon boundaries of CST6 in 11 patients with HI. No mutations were found in CST6, indicating it is not a major gene for types 1 and 2 HI. However, cystatin M/E protein expression was normal in patient tissues by immunohistochemistry, suggesting regulatory or noncoding mutations were unlikely. While CST6 does not appear to cause HI, further study of its role in skin differentiation and other skin disorders may provide insights into cornification pathways.
Genome sequencing uncovers MS phenocopies in primary progressive multiple scl...Sherman Jia
This document summarizes a study that performed whole-genome sequencing on patients with primary progressive multiple sclerosis (PPMS) and controls. The key findings were:
1) Some PPMS patients carried rare mutations in genes associated with neurological disorders that resemble MS, called phenocopies.
2) PPMS patients were enriched for rare mutations in genes that cause spastic paraplegias, a type of motor neuron disorder. This enrichment was unique to PPMS and not seen in relapsing-remitting MS.
3) Additional research is needed to understand how both rare and common genetic variations contribute to MS pathogenesis. The findings suggest PPMS may have features of both a complex genetic disease and monogenic
Oakes et al 2017 TBK1 - a new player in ALS linking autophagy and neuroinflam...Maria Davies
TBK1 is a kinase involved in innate immunity and autophagy pathways. Recent human genetics studies have linked mutations in TBK1 to amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons. TBK1 plays a major role in autophagy by phosphorylating autophagy adaptor proteins, and several other ALS-linked genes are also involved in autophagy. Mutations in TBK1 may impair autophagy and contribute to the accumulation of protein aggregates, a hallmark of ALS pathology. The review discusses the role of TBK1 in autophagy and how disrupted autophagy may underlie the pathogenesis of ALS
1) The study investigates how localized protein translation in axons regulates presynaptic development at synapses.
2) The researchers developed a method to selectively repress cap-dependent translation in axons using a targeted translational repressor.
3) They found that repressing axonal translation enlarged synaptic vesicle recycling pools, and this effect was partly due to decreased levels of p35, a protein involved in regulating vesicle recycling pools. Local translation of p35 mRNA in axons normally helps regulate vesicle recycling.
Als Paper & Prion Protein Gene Expressionjosephmdphysics
ALS is a degenerative neurological disease that causes motor neuron cell death leading to paralysis and death from respiratory failure. Free radicals, excess glutamate, calcium release, and genetic mutations contribute to motor neuron cell death. The disease progresses through corticomotor neuron death followed by spinal motor neuron denervation atrophy. Epidemiological studies show organophosphate pesticides and heavy metal exposures increase ALS risk by causing oxidative stress and gene imbalances that further damage cells. Electrophysiology shows abnormal cortical excitability occurs in sporadic ALS patients but not all genetic mutation cases, and excitability worsens with disease progression.
Motor neuron disease (MND) refers to conditions characterized by degeneration of upper and lower motor neurons. Amyotrophic lateral sclerosis (ALS) is the most common form of MND and involves both upper and lower motor neurons. ALS is defined clinically based on involvement of these motor neurons and leads to loss of muscle strength, atrophy, fasciculations, and difficulties with movement. The only approved treatment for ALS is riluzole, which may modestly extend survival by 2-3 months by reducing glutamate-induced excitotoxicity.
This document summarizes a project investigating whether mitochondrial DNA-encoded oxidative phosphorylation (OXPHOS) transcripts are dysregulated in the blood of patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared to controls. The student researcher designed primers, conducted quantitative real-time PCR on blood samples from controls and patients, and found several mtDNA-encoded OXPHOS transcripts were significantly more abundant in MCI and AD patients. This suggests peripheral changes in mitochondrial gene expression occur early in AD and could provide biomarkers for early diagnosis and monitoring disease progression and treatment responses.
Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
This document summarizes a study on developing a novel protein interaction platform to study neurodegeneration. The study cultured neuronal stem cells to form neurospheres, which were used as a model system. Lentiviral transfection was optimized to introduce target genes stably into the neurospheres. The goal was to analyze protein interactions of Alzheimer's disease proteins by co-immunoprecipitation of neurosphere cultures expressing tagged target proteins. This model aims to further the understanding of molecular mechanisms in neurodegenerative disorders like Alzheimer's disease.
1) The study characterized host vascularization of transplanted human neural stem cell grafts in porcine spinal cords. Twelve grafts of human neural progenitor cells were transplanted into three pigs' spinal cords.
2) Six weeks after transplantation, the pigs were sacrificed and the grafts were analyzed. Graft survival ranged from 0-65% and inversely correlated with host microvascular density in the grafts. More vascularized grafts had less cell survival.
3) Vessels in the grafts expressed vascular endothelial growth factor and contained proliferating endothelial cells and immune cells, which may impact graft survival. The findings could influence future immunosuppression regimens for cell transplantation.
Genetic Insights Into Multiple Sclerosis PathogenesisAaron Sparshott
A segment of a group presentation reflecting upon some of the genetic components that may contribute to Multiple Sclerosis pathogenesis.
IL2Rα and IL7Rα were the two genes of focus.
(This presentation was originally done for Semester 2 , 2008)
20140824 Abnormalities in human pluripotent cells due to reprogramming mechan...Alim Polat
This study compares genetic and epigenetic abnormalities in three types of human pluripotent stem cells: induced pluripotent stem cells (iPS cells), nuclear transfer embryonic stem cells (NT ES cells), and in vitro fertilization embryonic stem cells (IVF ES cells). The researchers found that both iPS cells and NT ES cells derived from the same somatic cells contained similar numbers of copy number variations, whereas DNA methylation and gene expression profiles of NT ES cells more closely matched those of IVF ES cells. The results suggest that human somatic cells can be faithfully reprogrammed to pluripotency by somatic cell nuclear transfer and may be better suited than iPS cells for cell replacement therapies due to avoiding
This research article describes the generation of new mouse models of partial trisomy and monosomy of human chromosome 21. The models contain extra or missing copies of a 9.4 Mb region on mouse chromosome 16 that is syntenic to a region on human chromosome 21. Mice with an extra copy (trisomy model) showed impaired locomotion and increased muscle strength/mass, while mice missing a copy (monosomy model) showed the opposite phenotype. Gene expression analysis revealed that genes related to muscle energy metabolism and mitochondria were downregulated in the trisomy mice and upregulated in the monosomy mice. This correlated with changes in mitochondrial proliferation and function in skeletal muscle, providing insight into the muscle and locom
1. The study investigated whether an axonal targeting sequence exists in the survival motor neuron (SMN) protein by analyzing a truncated form of SMN (SMN236) containing exons 2, 3 and 6.
2. The results showed that SMN236 co-localized with Gemin3 in axonal granules in neuroblastoma cells, similarly to full-length SMN. Granules containing SMN236 also exhibited bidirectional movement along axons comparable to granules containing full-length SMN.
3. These findings suggest that an axonal targeting sequence is located within exons 2, 3 and/or 6 of SMN. Identifying this sequence could help further characterize SMN's role
CODAS syndrome is a rare multi-system developmental disorder characterized by cerebral, ocular, dental, auricular, and skeletal anomalies. The researchers identified four mutations in the LONP1 gene in ten individuals with CODAS syndrome from three ancestral backgrounds. LONP1 encodes the mitochondrial Lon protease, which is involved in protein quality control and respiratory complex assembly in mitochondria. The mutations cluster in the AAA+ domain near the ATP-binding pocket and result in defects in ATP-dependent proteolysis. Lymphoblastoid cell lines from affected individuals show swollen mitochondria, aggregated cytochrome c oxidase subunit II, and reduced mitochondrial function, linking LONP1 mutations to CODAS syndrome.
This document discusses oligodendrocytes, the cells that form myelin in the central nervous system. It describes their role in development, diseases like multiple sclerosis where remyelination fails, and potential therapies to promote remyelination. A clinical trial was conducted using neural progenitor cell transplantation to treat Pelizaeus-Merzbacher disease, a fatal leukodystrophy caused by myelin deficiency. Research suggests the Wnt signaling pathway inhibits oligodendrocyte differentiation and myelination, and a small molecule Wnt inhibitor was found to accelerate remyelination in mice.
This study identified FUS, a gene frequently mutated in ALS, as autoregulating its own expression through alternative splicing of its pre-mRNA. The study found that FUS binds to and represses the splicing of its own exon 7. This causes the production of an exon 7-skipped splice variant that triggers nonsense-mediated decay, reducing FUS protein levels. Expression of ALS-linked FUS mutants compromised this autoregulation by reducing exon 7 repression and nuclear localization of FUS. This suggests deficient FUS autoregulation could exacerbate cytoplasmic accumulation of FUS in ALS through a feed-forward loop mechanism.
This article describes research identifying small molecule pan-caspase inhibitors that could potentially treat Huntington's disease. The researchers screened compounds against caspase-3 and -6 to identify three novel inhibitors that blocked huntingtin protein cleavage by these caspases. Testing in Huntington's disease models found that the irreversible inhibitors suppressed toxicity from mutant huntingtin protein and rescued neurons from cell death. The results support that caspase-mediated cleavage of huntingtin plays a critical role in Huntington's disease pathogenesis, and inhibiting caspases may be a promising therapeutic strategy.
This document reports on a study that tested the effects of isoxazole 9 (Isx-9), a small synthetic molecule, on adult hippocampal neurogenesis in rats. The study found that administering Isx-9 for 14 days potentiated cell proliferation and increased the number of immature neurons in the hippocampal dentate gyrus. Isx-9 treatment also completely reversed the reduction in cell proliferation and neuronal commitment observed in vehicle-treated animals that were subjected to repeated handling and injections. These findings demonstrate that Isx-9 has promising pro-neurogenic properties and could help mitigate stress-induced deficits in adult hippocampal neurogenesis.
Mitochondrial diseases are caused by defects in mitochondrial structure or enzymes that result in deficient energy production. They can affect multiple organ systems and occur across all age groups. Mitochondrial DNA mutations can be inherited from the mother and nuclear DNA mutations can affect mitochondrial proteins or DNA maintenance. Common mitochondrial diseases include MELAS, MERRF, and Leigh syndrome. Mitochondrial dysfunction has also been implicated in aging and common diseases like heart disease and Parkinson's.
This study used an AAV vector to express clarin-1 (CLRN1) in the mouse retina to determine its subcellular localization. CLRN1 was expressed in all major retinal cell types when driven by a ubiquitous promoter. In photoreceptors, CLRN1 localized mainly to the inner segment and outer plexiform layer, similar to other usher proteins. High-titer subretinal delivery led to loss of retinal function, suggesting a critical limit for CLRN1 expression. The results imply CLRN1 expression may be supported by multiple retinal cells and that the dose, promoter, and delivery method need optimization for USH3 gene therapy.
1) Mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 (NL2) were found to exhibit increased anxiety-like behavior.
2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
This study aimed to determine if mutations in the human cystatin M/E gene (CST6) contribute to harlequin ichthyosis (HI) by sequencing the entire coding region and intron-exon boundaries of CST6 in 11 patients with HI. No mutations were found in CST6, indicating it is not a major gene for types 1 and 2 HI. However, cystatin M/E protein expression was normal in patient tissues by immunohistochemistry, suggesting regulatory or noncoding mutations were unlikely. While CST6 does not appear to cause HI, further study of its role in skin differentiation and other skin disorders may provide insights into cornification pathways.
Genome sequencing uncovers MS phenocopies in primary progressive multiple scl...Sherman Jia
This document summarizes a study that performed whole-genome sequencing on patients with primary progressive multiple sclerosis (PPMS) and controls. The key findings were:
1) Some PPMS patients carried rare mutations in genes associated with neurological disorders that resemble MS, called phenocopies.
2) PPMS patients were enriched for rare mutations in genes that cause spastic paraplegias, a type of motor neuron disorder. This enrichment was unique to PPMS and not seen in relapsing-remitting MS.
3) Additional research is needed to understand how both rare and common genetic variations contribute to MS pathogenesis. The findings suggest PPMS may have features of both a complex genetic disease and monogenic
Oakes et al 2017 TBK1 - a new player in ALS linking autophagy and neuroinflam...Maria Davies
TBK1 is a kinase involved in innate immunity and autophagy pathways. Recent human genetics studies have linked mutations in TBK1 to amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons. TBK1 plays a major role in autophagy by phosphorylating autophagy adaptor proteins, and several other ALS-linked genes are also involved in autophagy. Mutations in TBK1 may impair autophagy and contribute to the accumulation of protein aggregates, a hallmark of ALS pathology. The review discusses the role of TBK1 in autophagy and how disrupted autophagy may underlie the pathogenesis of ALS
1) The study investigates how localized protein translation in axons regulates presynaptic development at synapses.
2) The researchers developed a method to selectively repress cap-dependent translation in axons using a targeted translational repressor.
3) They found that repressing axonal translation enlarged synaptic vesicle recycling pools, and this effect was partly due to decreased levels of p35, a protein involved in regulating vesicle recycling pools. Local translation of p35 mRNA in axons normally helps regulate vesicle recycling.
Als Paper & Prion Protein Gene Expressionjosephmdphysics
ALS is a degenerative neurological disease that causes motor neuron cell death leading to paralysis and death from respiratory failure. Free radicals, excess glutamate, calcium release, and genetic mutations contribute to motor neuron cell death. The disease progresses through corticomotor neuron death followed by spinal motor neuron denervation atrophy. Epidemiological studies show organophosphate pesticides and heavy metal exposures increase ALS risk by causing oxidative stress and gene imbalances that further damage cells. Electrophysiology shows abnormal cortical excitability occurs in sporadic ALS patients but not all genetic mutation cases, and excitability worsens with disease progression.
Motor neuron disease (MND) refers to conditions characterized by degeneration of upper and lower motor neurons. Amyotrophic lateral sclerosis (ALS) is the most common form of MND and involves both upper and lower motor neurons. ALS is defined clinically based on involvement of these motor neurons and leads to loss of muscle strength, atrophy, fasciculations, and difficulties with movement. The only approved treatment for ALS is riluzole, which may modestly extend survival by 2-3 months by reducing glutamate-induced excitotoxicity.
1) ALS is a progressive neurodegenerative disease that affects motor neurons, leading to muscle paralysis. It was first described in 1874 but its pathogenesis remains unclear.
2) ALS may be caused by both increased toxicity from substances like RNA proteins and weakness of the motor neuron system. Genetic mutations and environmental toxins are also implicated in some cases.
3) The document proposes new experimental and drug delivery approaches to better understand disease mechanisms and improve treatment of ALS.
Motor neuron disease (MND) refers to conditions characterized by degeneration of upper and lower motor neurons. Amyotrophic lateral sclerosis (ALS) is the most common form of MND and involves both upper and lower motor neurons. ALS is clinically defined based on involvement of motor neurons and includes features such as muscle weakness, atrophy, fasciculations, and stiffness. The pathology of ALS involves degeneration and death of motor neurons in the brain, brainstem, and spinal cord leading to muscle denervation and atrophy. While the cause of ALS is largely unknown, factors such as oxidative stress, protein aggregation, mitochondrial dysfunction, and glutamate excitotoxicity are hypothesized to contribute to motor neuron de
The Main Molecular Bases of Amyotrophic Lateral Sclerosis: An Integrating Reviewasclepiuspdfs
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that affects the lower and upper motor neurons. Its etiology is unknown, despite several studies that point to risk factors and genetic for the disease. It does not have a definitive treatment and the diagnosis, most of the time, is time consuming and demands varied criteria for adequate definition and classification. In the search for new treatments, gene therapy with gene insertion aims to reduce the impact of ALS on the patient. The study of molecular basis as the root cause of the disease becomes necessary to advance the discovery of effective treatment and a rapid diagnosis, aiming at improving the quality of life of the ALS patients. Therefore, the present work had the objective to bring an update of the literature on ALS, as well as to describe the main molecular bases involved in the causative/causative process of this disease.
Spines, plasticity, and cognition in alzheimer model mice.shiraknafo
This review article discusses research on transgenic Alzheimer's disease (AD) mouse models and how they are used to investigate dendritic spine structure, synaptic function, and cognition. Studies show that soluble amyloid-beta initiates synaptic dysfunction and loss, as well as tau pathology, contributing to both synaptic and neuronal loss. These changes in synapse structure and function, as well as outright synapse and neuron loss, underlie the neural system dysfunction that causes cognitive deficits in AD. Understanding how dementia develops in AD is crucial for developing effective therapies.
This article reviews studies on transgenic Alzheimer's disease (AD) mouse models that investigate dendritic spine structure, synaptic function, and cognition. These studies show that soluble amyloid-beta initiates synaptic dysfunction and loss, as well as pathological changes in tau, which contribute to both synaptic and neuronal loss. In vivo imaging reveals plaque-associated spine loss and structural plasticity deficits in AD mouse models. Synaptic plasticity is also severely impaired in AD mouse models, with deficits in basal synaptic transmission and long-term potentiation emerging with age. Together, these changes in synapse structure and function are thought to underlie the cognitive deficits observed in AD.
Genes in complex neurological disordersDenise Sheer
The document discusses approaches for identifying genes associated with neurological disorders. It describes several methods: linkage studies, homozygosity mapping, exome and whole genome sequencing, genome-wide association studies, detection of structural variation, and transcriptomics. Each method is summarized, with examples provided of neurological genes identified through various approaches such as exome sequencing. The human genome is also briefly described to provide context. In summary, the document outlines strategies for pinpointing genes underlying neurological conditions.
A mitochondrion (singular of mitochondria) is part of every cell in the body that contains genetic material.
Mitochondria are responsible for processing oxygen and converting substances from the foods we eat into energy for essential cell functions.
The mitochondria of the zygote come from the oocyte, that is, from the mother and almost never from the sperm, form of transmission is called maternal inheritance
Which mitochondrial gene is mutated.
The extent of replicative segregation of the mutant mitochondrial genome during the early stages of embryonic development.
The abundance of the mutant mitochondrial gene in a particular tissue.
The threshold level of mutant mitochondrial DNA required in a tissue before an abnormality is evident clinically
Mitochondrial disease affects tissues most highly dependent on ATP production
*Nerves
*Muscles
Endocrine
Kidney
Low energy-requiring tissues are rarely directly affected, but may be secondarily
Lung
Connective tissue
Symptoms can be intermittent
Increased energy demand (illness, exercise)
Decreased energy supply (fasting)
Common feature
myoclonus epilepsy, deafness, blindness, anemia, diabetes, seizures and loss of cerebral blood supply (stroke).
Myoclonic epilepsy and ragged-red fiber disease (MERRF)
MERRF is a member of a group of disorders called mitochondrial encephalomyopathies that feature mitochondrial defects with altered brain and muscle functions.
The term “ragged red fibers” refers to large clumps of abnormal mitochondria that accumulate mostly in muscle cells and are stained red by a dye that is specific for complex II of the electron transport chain.
rare, maternally inherited, heteroplasmic, (point mutation in tRNA lysine gene)
Mutation is MTTK*MERRF8344G.
MT means mitochondrial gene is mutated
T means transfer RNA gene
K means the single-letter amino acid designation for lysine
MERRF means the clinical features
8344G means the mutant nucleotide is guanine (G) at nucleotide position 8344
If 90% of the mitochondria in nerve and muscle cells carry the MTTK*MERRF8344G mutation, then the defining symptoms of MERRF are present.
Maternally inherited mitochondrial disease
The MTTL1*MELAS3243G mutation accounts for more than 80% of the cases of MELAS.
This base substitution is in one of the two mitochondrial transfer RNALeu genes.
the A3243G mutation occurs in thetRNALeu(UUR) gene
When this mutation is present in ≥90% of the mitochondrial DNA of muscle tissue, there is an increased likelihood of recurrent strokes, dementia, epilepsy, and ataxia.
When heteroplasmy for the A3243G mutation
is ~40% to 50%, chronic progressive external ophthalmoplegia (CPEO), myopathy, and deafness are likely to occur.
Other MELAS mutations occur at sites 3252, 3271, and 3291 within the tRNALeu(UUR) gene and in the mitochondrial tRNAVal (MTTV) and COX III (MTCO3) genes.
Reduced activities in Complexes I and IV are established
Amyotrophic lateral sclerosis (ALS) is a rare neurological disease that primarily affects the nerve cells (neurons) responsible for controlling voluntary muscle movement (those muscles we choose to move). Voluntary muscles produce movements like chewing, walking, and talking.
This document discusses perspectives in molecular imaging and immunotherapies for Alzheimer's disease (AD). It provides background on AD, reviewing current theories for its pathogenesis including the cholinergic, amyloid-beta, and tau hypotheses. Imaging technologies like positron emission tomography (PET) and magnetic resonance imaging (MRI) can help with early diagnosis by detecting biomarkers for different stages of AD in cerebrospinal fluid and brain tissue. Combining molecular imaging systems may improve accuracy. Current and future immunotherapies aim to treat or prevent AD by targeting its underlying molecular mechanisms.
This document summarizes research on amyotrophic lateral sclerosis (ALS), including its pathogenesis, genetic factors, and potential new treatment strategies. It discusses how ALS results from damage to motor neurons in the brain and spinal cord from a combination of increased toxicity from substances like glutamate, calcium ions, and protein aggregates as well as weaknesses in the motor neuron system from genetic mutations and deficits in neurotrophic factors. While the cause is still unclear, various studies suggest roles for inflammation, immune responses, oxidative stress, cytoskeletal abnormalities, and dysregulation of autophagy and microRNAs. The document reviews several studies examining these pathways and their implications for understanding ALS and identifying new therapeutic targets. It also proposes experimental models to
Radial sorting allows Schwann cells to segregate axons and is required for myelination in the peripheral nervous system. This process is impaired in mouse models of human diseases caused by mutations in laminin α2, Large, or fukutin genes. These genes are involved in laminin signaling or glycosylation. It is unclear which receptor(s) mediate laminin function during sorting. Candidates are β1 integrins, but their phenotype differs from laminin/glycosyltransferase mutants. Deletion of the Large/fukutin substrate dystroglycan alone does not affect sorting. This study shows that removing dystroglycan in a specific genetic background causes sorting defects matching laminin mutants, recap
This document summarizes a book titled "Role of Plants, environmental toxins and physical neurotoxicological factors in Amyotrophic lateral sclerosis, Alzheimer Disease and Other Neurodegenerative Diseases." It has 10 authors from various countries. The book aims to observe the effects of plant neurotoxins, physical factors, and geography on neurodegenerative diseases like ALS, PD, and AD. It reviews literature on geographic clusters of these diseases and genetic mutations. It also discusses the brain's high metabolic needs, interactions between neurons and astrocytes, and how impairments in these systems can lead to neurodegeneration. The book presents figures and intends to develop new hypotheses and therapies based on contributing pathogenic factors.
This study establishes an in vitro model of primary muscle fibers derived from SOD1G93A mice that recapitulates features of amyotrophic lateral sclerosis (ALS) seen in vivo. The model shows impaired muscle development and neuromuscular junction maintenance in SOD1G93A fibers compared to wild-type fibers. Specifically, SOD1G93A fibers have fewer acetylcholine receptor clusters, reduced expression of proteins important for cluster formation like MuSK and rapsyn, thinner fibers, and fewer triads. Analysis of isolated fibers from symptomatic SOD1G93A mice found similar neuromuscular impairments, validating the in vitro model. This novel model supports the hypothesis that impaired muscle development
This document discusses the role of various factors in neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease. It notes that geographic clusters of ALS have been observed in areas such as Guam and Japan. It also discusses the metabolic cooperation between neurons and astrocytes in the brain, highlighting how disruptions to this interaction can lead to neurodegenerative disorders. The document reviews literature on various pathogenic mechanisms and potential environmental and genetic risk factors involved in these conditions.
The document reports on a study investigating the molecular mechanisms underlying epilepsy associated with tuberous sclerosis complex (TSC) using a mouse model. The key findings are:
1) Heterozygous Tsc1+/- mice exhibited spontaneous seizures during early postnatal life despite lacking major brain malformations, suggesting haploinsufficiency contributes to the epileptic phenotype.
2) Seizures in Tsc1+/- mice originated in the granular layer (L4) of the neocortex before spreading to other layers.
3) GluN2C-containing NMDA receptors were functionally upregulated in the neocortex of Tsc1+/- mice in an mTOR-dependent manner
1) The researchers found a statistically significant decrease in the levels of the glutamatergic synaptic markers VGLUT1 and PSD-95 in the striatum of mutant Huntington's disease mouse models compared to wild-type mice, using immunohistochemistry and western blotting techniques.
2) Astrogliosis, a marker of synaptic deterioration, was also observed qualitatively and quantitatively increased in the striatum of mutant mice.
3) The results suggest that mutant mice exhibit a reduced number of corticostriatal glutamatergic synapses, potentially preceding neuronal cell loss in Huntington's disease.
The prevalence of Copy Number Variation (CNV) on human chromosome 16p11.2, identified in approximately 1% of autism spectrum disorder (ASD) cases globally, was found to be 1.2% in an Australian ASD cohort. Bioinformatic analysis identified 13 of the 25 protein coding genes within the 16p11.2 region, including KCTD13, as significantly enriched in the nervous system. Experiments in mice found that suppression of Kctd13 led to impaired radial migration of cortical neurons and altered neuronal morphology, providing insight into how perturbations to KCTD13 expression via 16p11.2 microdeletion could influence brain development. Further experiments showed decreased cell proliferation and mitosis levels
This document summarizes a study examining the relationship between impaired brain insulin signaling, amyloid-beta oligomers (AbOs), tau, and cell cycle reentry (CCR) in Alzheimer's disease (AD) pathogenesis. The study finds that AbOs activate the protein kinase mTORC1, which then phosphorylates tau and induces CCR in neurons. CCR can be prevented by insulin-stimulated activation of lysosomal mTORC1. As AbOs also reduce neuronal insulin signaling, decreased insulin signaling allows the toxic effects of AbOs to cause CCR and neuronal death, contributing to AD progression. The findings help explain how impaired brain insulin signaling may promote AD by unleashing the cell cycle reentry effects of Ab
Similar to Scientifi c Journal of Multiple Sclerosis (20)
A 5-year old boy, with an established diagnosis of a topic
dermatitis, previously treated by topical corticosteroids and emollient cream with a good improvement, developed widespread papules on his legs, hands and forearm that appeared 5 months ago.
Methods: Retrospectively, the file records of the patients who underwent sleeve gastrectomy were examined. Demographic features, Body Mass Index (BMI), the mouth opening, Mallampati score, thyromental distance, sternomental distance, neck circumference measurements and videolaryngoscopic examination results were recorded Results: In a total of 140 consecutive patients (58 male, 82 female) were included in the study. The mean age of the study participants was 35.40 ± 9.78 and the mean BMI of the patients was 44.33 ± 7.52 kg/m2
. The mean mouth opening of the patients was 4.82 ± 0.54 cm
and the mean neck circumference was 43.52 ± 4.66 cm. The mean thyromental distance was 8.02 ± 1.00 cm and the mean sternomental distance was16.58 ± 1.53 cm. Difficult intubation was determined in 8 (5.7%) patients. In logistic regression analysis, age (p : 0.446), gender (p : 0.371), BMI (p : 0.947), snoring (p : 0.567), sleep apnea (p : 0.218), mouth opening (p : 0.687), thyromental distance (p :0.557), sternomental (p : 0.596) and neck circumference (p : 0.838) were not the independent predictors of difficult intubation. However, Mallampati score (p : 0.001) and preoperative direct laryngoscopy findings (p : 0.037) performed in outpatient clinic were the significant
predictors of difficult intubation. Interestingly, all patients with grade 4 laryngoscopy findings had difficult intubation.
Introduction: Laparoscopic surgery has been performed in Mexico since 1989, but no reports about training tendencies exist. We conducted a national survey in 2015, and here we report the results concerning training characteristics during the surgical residence of the respondents. Materials and Methods: A prospective study was conducted through a survey questioning demographic data, laparoscopic training during pre and post surgical residency and other of areas of laparoscopic practice. The sample was calculated and survey piloted before
application. Special interest in this report was placed on type and quality of training received. Data are reported in percentages.
Heterotopic Ossification (HO) is defined as pathological bone formation at locations where bone normally does not exist. The
presence of HO has been found to be a rare complication after stroke in several studies, whereas there are only sporadic references relating HO to Cerebral Palsy (CP) and few for CP and stroke. No effective treatment for HO has yet been found, whereas the cellular and molecular mechanisms have not been completely understood. Therefore, increased awareness among physicians is required, as a challenge for early diagnosis and treatment. A case of a male patient with CP, who developed HO on the paretichip joint following an ischemic stroke is presented.
Objectives: To assess the practice of food hygiene and safety, and its associated factors among street food vendors in urban areas of Shashemane, West Arsi Zone, Oromia Ethiopia, 2019.
Methods: Cross-sectional study design was applied from December 28, 2019 to January 27, 2020. Data was collected from 120 food handlers, which were selected by purposive sampling techniques. Information was gathered from interview and field observation by conducting food safety survey and using questionnaires via face to face interview. The collected data was entered using Epi Data 3.1 and finally, it was analyzed using SPSS VERSION 20.
A Division I football player experienced acute posterior leg pain while playing. An ultrasound examination revealed an unusual injury - a complete rupture of the plantaris tendon mid-substance. This type of isolated plantaris tendon injury has rarely been reported. Ultrasound was useful for diagnosis and guided rehabilitation by monitoring healing over time. The athlete was able to return to full competition within 3 weeks through a progressive rehabilitation program focused on restoring range of motion and strength. This case suggests isolated plantaris tendon injuries may allow for faster return to play than other potential causes of posterior leg pain.
Type 1 Diabetes (T1D), is a severe disease, representing 5-10% of all reported cases of diabetes worldwide. Fulminant Type 1 Diabetes Mellitus (FT1D) is a subtype of type 1 diabetes mellitus that is largely characterized by the abrupt onset of Diabetic Ketoacidosis (DKA) and severe hyperglycemia without insulin defi ciency. Viral infections have been hypothesized to play a major role in the pathogenesis of Fulminant Type 1 Diabetes Mellitus (FT1D) through the complete and rapid destruction of pancreatic beta cells. Coxsackie viral infection has been detected in islets of 50% of the pancreatic tissue recovered from recent-onset Type 1 Diabetes (T1D) patients. In this report we have highlighted a case where the patient developed a Group B Coxsackie virus infection culminating in the development of Fulminant Type 1 Diabetes Mellitus (FT1D).
Methods: Cercariae are released by infected water snails. To determine the occurrence of cercariae-emitting snails in SchleswigHolstein, 155 public bathing places were visited and searched for fresh water snails. Family and genus of the collected snails were determined and the snails were examined for the shedding of cercariae, using a standard method and a newly developed method.
Objective: To generate preliminary information about of enteroviruses and Enterovirus 71 (EV71) in patients with aseptic meningitis in Khartoum State, Sudan.
Method: Cerebrospinal fluid specimens were collected from 89 aseptic meningitis patients from different Khartoum Hospitals
(Mohammed Alamin Hamid Hospital, Soba Teaching Hospital, Omdurman Military Hospital, Alban Gadeed Teaching Hospital and Police Hospital) within February to May 2015. Among these 89 patients, 43 (48%) were males and 46 (52%) were females. The patient’s age ranged between 1 day and 30 years old. The collected specimens were assayed to detect enteroviruses and EV71 RNA using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique
Femoral hernias, comprise 2% to 4% of all hernias in the inguinal region, and occur most commonly in women. Th ey present typically with a mass below the level of the inguinal ligament. The sac may contain preperitoneal fat, omentum, small bowel, or other structures and have a high rate of incarceration and strangulation due to the small size of the hernia neck orifice, requiring emergency surgery. We present the case of a 54-year-old female patient with intestinal occlusion due to incarcerated femoral hernia, repaired by laparoscopic approach, that gave the patient the opportunity to attend her daughter’s wedding the same day.
Small Supernumerary Marker Chromosome (sSMC) is a rare genetic condition marked by the presence of an extra chromosome to the 46 human chromosomes. This case report describes a 4 year old child with SSMC on the 46th chromosome. The child presented with delayed speech and language development, seizures and mild developmental delay. Speech and Language evaluation was carried out and management options are discussed.
A catheter is a thin tube made from medical grade materials that serve a broad range of functions, but mainly catheters are medical devices that can be inserted in the body to treat disease or perform surgical procedures. Catheters have been inserted into body cavities, ducts, or vessels to allow for drainage, administration of therapeutic fluids or gases, operational access for surgery. Catheters help perform tasks in various systems such as cardiovascular, urological, gastrointestinal, neurovascular, and ophthalmic systems. A dataset of 12 patients with varying “weights” and “heights” was recorded along with the lengths of their catheter tubes. This data set was found from two revered statistical textbooks on linear regression and the Department of Scientific Computing at Florida State University. This data set was not able to be linked to any particular clinical or experimental research studies, but the data set can be used to help catheter manufacturers and medical professionals better decide on what particular catheter lengths to use for patients knowing only their height & weight. These research insights could be helpful to healthcare professionals that have patients with incomplete or no healthcare records
to decide what catheter length to use. The main investigative inquiry that needed to be answered was how does patient weight & height influence catheter length together and separately? We conducted linear regression and other statistical analysis procedures in R program & Microsoft Excel and discovered that this data exhibited a quality called multi collinearity. With multi collinearity, all predictors (2 or more
independent variables) are not significant in an all encompassing linear aggression, but the predictors might be significant in their own individual linear regressions. Individual linear regression analyses were conducted for both patient height & weight to see how much they both contribute to varying catheter length. Patient weight was found to be more impatful than patient height in relationship to catheter length, even though height and weight are a classical example of multi collinearity predictors.
Bovine mastitis has a negative impact through economic losses in the dairy sector across the globe. A cross sectional study was carried out from September 2015 to July 2016 to determine the prevalence of bovine mastitis, associated risk factors and isolation of major causative bacteria in lactating dairy cows in selected districts of central highland of Ethiopia. A total of 304 lactating cows selected randomly from five districts were screened by California Mastitis Test (CMT) for subclinical mastitis. Based on CMT result and clinical examination, over all prevalence of mastitis at cow level was 70.62% (214/304).
Two hundred fourteen milk samples collected from CMT positive cows were cultured for isolation of major causative bacteria. From 214 milk samples,187 were culture positive and the most prevalent isolates were Staphylococcus aureus 42.25% (79/187) followed by Streptococcus agalactiae 14.43%
(27/187). Other bacterial isolates were included Coagulase Negative Staphylococcus species 12.83% (24/187), Streptococcus dysgalactiae 5.88% (11/187), Escherichia coli 13.38% (25/187) and Entrococcus feacalis 11.23% (21/187) were also isolated. Moreover, age, parity number, visible teat abnormalities,husbandry practice, barn fl oor status and milking hygiene were considered as risk factors for the occurrence of bovine mastitis and they were found significantly associated with the occurrence of mastitis (p < 0.05). The findings of this study warrants the need for strategic approach including dairy extension that focus on enhancing dairy farmers’ awareness and practice of hygienic milking, regular screening for subclinical mastitis, dry cow therapy and culling of chronically infected cows.
A 36-year-old female developed right upper quadrant pain and nausea after taking the herbal supplement kratom for two weeks to manage back pain. Laboratory tests showed elevated liver enzymes. A liver biopsy ruled out other causes and determined she had drug-induced liver injury from kratom use. Her symptoms and liver enzymes gradually returned to normal over six weeks after stopping kratom. The case report discusses kratom's potential for hepatotoxicity and advises clinicians to consider its effects on patient health.
The assessment, diagnosis and treatment of critically ill patients is extremely challenging. Patients often deteriorate whilst being
reviewed and their rapidly changing pathophysiology barrages healthcare professionals with new data. Furthermore, comprehensive assessments must be postponed until the patient has been stabilised. So, important data and interventions are often missed in the heat of the moment. In emergency situations, suboptimal management decisions may cause signifi cant morbidity and mortality. Fortunately, standardisation and careful design of documentation (i.e. proformas and checklists) can enhance patient safety. So, I have developed a series of checklist proformas to guide the assessment of critically ill patients. These proformas also promote the systematic recording and presentation of information to facilitate the retrieval of the precise data required for the management for critically ill patients. The proformas have been modifi ed extensively over the last twenty years based on my personal experience and extensive consultation with colleagues in several world-renowned centres of excellence. The proformas were originally developed for use in the intensive therapy unit
or high dependency unit. However, they have been adapted for use by outreach teams reviewing patients admitted outside of critical care areas. The use of these tools can direct eff orts to provide appropriate organ support and provides a framework for diagnostic reasoning.
This review article discusses microvascular and macrovascular disease in systemic hypertension. It summarizes that:
1) Cardiac imaging plays a crucial role in risk stratifying hypertensive patients and identifying management strategies by properly diagnosing microvascular and coronary artery disease.
2) The nitric oxide synthase (eNOS) G298 gene allele may be a marker for microvascular angina in hypertensive patients, as studies have found it to be more prevalent in hypertensive patients with chest pain and reversible myocardial defects but normal coronary arteries.
3) Both structural changes like capillary rarefaction and functional changes like endothelial dysfunction can cause microvascular dysfunction and angina in hypertensive individuals in the absence of
This study characterized dengue infections in Pakistan by analyzing hematological and serological markers in 154 suspected dengue cases and 146 control patients with other febrile illnesses. NS1 antigen was detected in 55% of dengue cases, IgM antibodies in 30%, and both in 15%. Control groups primarily had malaria (71%) and enteric fever (20%). Hematological markers (platelet count, hematocrit, WBC) measured before and after treatment showed significant differences for platelet count and hematocrit but not WBC count between the groups. Analysis of clinical symptoms and serological/hematological markers helps diagnose dengue, assess prognosis, and inform prevention efforts to reduce morbidity, mortality and spread of the disease.
Researchers from Utrecht recently published yet another paper on the use of Magnetic Resonance Imaging (MRI)demonstrating an additional failed attempt to understand the importance of qualitative versus quantitative imaging, and anatomic versus physiologic imaging. Th e implications of this failure here cannot be overstated.
Introduction: Stroke is an even more dramatic major public health problem in young people. Goal of the study: Contribute to the knowledge of strokes in young people. Methodology: This was a retrospective study carried out over a period of 02 years (January 2017 to December 2018) including the files of patients aged 18 to 49 years hospitalized for any suspected case of stroke in the Neurology department of the University Hospital
Center of the Sino-Central African Friendship (CHUSCA) of Bangui.
Background: This report describes a unique case of a patient that developed psychotic symptoms believed to be secondary
to a tentorial meningioma with associated hydrocephalus. These psychotic symptoms subsequently abated with placement of a
ventriculoperitoneal shunt. Case description: 60-year-old female was admitted to an inpatient psychiatric facility on a psychiatric involuntary commitment petition due to progressive paranoia, homicidal ideation and psychosis. The work up showed a calcified six cm tentorial meningioma with associated hydrocephalus. The patient initially rejected treatment but later became amenable to placement of Ventriculoperitoneal Shunt
(VPS).
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2. Scientific Journal of Multiple Sclerosis
SCIRES Literature - Volume 2 Issue 1 - www.scireslit.com Page -002
ABBREVIATIONS
AmyotrophicLateralSclerosis-ALS;FamilialALS-fALS;Analysis
Of Variance - ANOVA; Choline Acetyltransferase - ChAT; Cu, Zn-
Superoxide Dismutase - SOD1; Mutant SOD1 - mSOD1; Normal
Control - Nc; Hydroxyl Radical - .
OH; Neuron Specific Enolase - NSE;
Peroxynitrite - ONOO-
; Superoxide Anion - O2
-
; Phosphate-Buffered
Saline - PBS; Reactive Species - RS; Transgenic - Tg; Transmission
Electron Microscopy - TEM; Terminal Deoxynucleotidyl Transferase
(TdT) Mediated Deoxyuridine Triphosphate-Biotin Nick End
Labeling - TUNEL; Enzyme-Linked Immuno-Sorbent Assay- ELISA
INTRODUCTION
Amyotrophic Lateral Sclerosis (ALS) is a progressive
degenerative disorder of motoneurons in the spinal cord, brain
stem and motor cortex. The disease typically strikes adults, usually
causing paralysis and death after five years of symptom onset. A
number of mechanisms have been proposed for the pathogenesis of
ALS, including genetic factors, oxidative stress, abnormal protein
modifications, protein misfolding and aggregation, misregulation of
RNA transcription, excess excitotoxicity of glutamate, mitochondrial
dysfunction, impaired axonal transport, inflammatory cascades,
dysfunctional signaling pathways, and apoptotic cell death. Despite
extensive research, the mechanisms of ALS pathogenesis remain
obscure, and no effective therapy is available [1-5]. Approximately
10% of ALS cases are inherited. The discovery of mutations in the
Cu, Zn-superoxide dismutase (SOD1) gene in familial ALS (fALS)
patients [6] was the first breakthrough towards identifying the cause
of fALS. Based on this discovery, a Transgenic (Tg) mouse model was
established by introducing a mutant human SOD1 gene (Gly 93 →
Ala, G93A) into the mouse [7]. These Tg mice developed symptoms
resembling human ALS. Although over 100 different SOD1 mutations
have been identified in fALS families and a number of Tg mouse
models with different mutation sites have been developed [8], most
of the research addressing ALS pathogenesis and the development
of therapeutic strategies has been performed using the G93A mutant
SOD1 (mSOD1) Tg model [9-19]. Therefore, the G93A mSOD1 Tg
mouse is still a very powerful and popular animal model for studying
the pathogenesis of ALS. The hereditary and non-hereditary forms of
the disease are clinically indistinguishable; thus, the final nerve cell
destruction is likely the same for both. Information obtained using
the mSOD1 mouse model should therefore also be informative to the
much larger number of cases of sporadic, non-inherited ALS. In the
present study, the most popular G93A mSOD1 Tg mouse model was
used and the results compared with those in wild-type SOD1 (SOD1)
mouse and normal mouse (Nc) without gene transfection.
Studies using genetically engineered mouse models have indicated
that the expression of mutant SOD1 in neurons alone is insufficient
to cause degeneration of motoneurons if they are surrounded
by a sufficient number of normal non-neuronal cells [8,20-22].
Conversely, wild type/normal- or mSOD1-deleted motoneurons,
surrounded by mSOD1-containing non-neuronal cells lead to signs
of abnormality. Therefore, the participation of non-neuronal cells
such as microglia [23,24], astrocytes [10,25- 27] and oligodendrocytes
[28] may contribute to the pathogenesis of the ALS disease [15,29].
These findings changed the concept of ALS as solely a motoneuron
degeneration disorder to a complex multi systemic and multifactorial
syndrome in which motoneuron degeneration is only part of a wider
multifaceted disease process. However, opposite report exist: selective
insertion of the G93A SOD1 mutant into motoneurons has shown
that these cells inevitably undergo degeneration and death, regardless
of the status of the surrounding glial cells [30].Therefore, it is
important to further explore the roles of surrounding non-neuronal
cells in this motoneuron degenerative disease.
Apoptosis has been suggested to be one of the pathways
responsible for motoneuron death. Apoptosis is important in
regulating normal development and maintaining tissue homeostasis
in the adult. However, too much or too little apoptosis can result in
pathological disorders [31,32]. Molecular evidence from animal and
cellular models as well as postmortem tissues indicates that apoptosis
occurs in motoneuron death in ALS [11-14,33-36]. However, the
morphological evidence for apoptosis in ALS remains equivocal and
contradictory [9,12,37]. Therefore, the present study is to provide
sound in vivo evidence of apoptosis in motoneuron and surrounding
non-neuronal cells so as to clarify the role of apoptosis in ALS.
To explore the role of apoptosis in ALS, DNA fragmentation
was examined as a biochemical marker of apoptosis by fluorescence
ABSTRACT
Amyotrophic Lateral Sclerosis (ALS) is a progressive motoneuron degenerative disease; its cause and mechanisms of pathogenesis
remain obscure. Apoptosis may be one of the pathways responsible for motoneuron death in ALS. The present study is to further explore
the role of apoptosis in ALS. Using a mutant Cu, Zn-superoxide dismutase (mSOD1) transgenic mouse model, we investigated the
correlation between SOD1 mutation and neuronal and glial apoptosis in different brain regions. TUNEL-fluorescence staining and ELISA
quantification found significantly more DNA fragmentation in mSOD1 mice than in controls. Double staining with TUNEL + an anti-choline
acetyltransferase antibody showed many TUNEL-positive motoneurons and glial cells in the spinal cord and brain stem of mSOD1 mice,
but not in the controls. Transmission electron microscopy confirmed neuronal and glial apoptosis in the spinal cord, brain stem, and
motor cortex by specific morphological features of apoptosis. Double staining with TUNEL + an anti-neuron-specific enolase antibody
showed many TUNEL-positive neurons in both the motor and sensory cortices of mSOD1 mice, but not in the controls. Counting the
number of TUNEL-positive neurons in the sections from the three mouse groups showed significantly more TUNEL-positive neurons in
both the motor and sensory cortices of mSOD1 mice than in the corresponding regions of control mice. There is no significant difference
between motor and sensory cortices of mSOD1 mice. These results provide firm in vivo evidence that SOD1 mutation induced apoptosis
of motoneurons and non-motoneuron cells in motoneuron-enriched brain regions. Therefore, apoptosis is not specific for motoneuron
death, but a common pathway causing neuronal and glial degeneration and death in motoneuron-enriched brain regions. SOD1 mutation
also induced apoptosis in sensory neurons of sensory cortex. Together, these findings imply that apoptosis might be one of pathways in
which the non-neuronal cells and non-motoneuron-enriched brain regions involve in motoneuron death in ALS.
Keywords: Amyotrophic Lateral Sclerosis; Cu, Zn-Superoxide Dismutase Mutation; Neuronal And Glial Apoptosis; Motoneuron
Degeneration; Transgenic Mouse Model; Transmission Electron Microscopy
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staining of Terminal Deoxynucleotidyl Transferase (TdT)-mediated
deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-
positive nuclei and by enzyme-linked immuno-sorbent assay (ELISA)
in the three groups of mice to determine whether mSOD1 mice are
more susceptible to DNA fragmentation than controls are. TUNEL-
positive neurons (TUNEL + neuron-specific enolase, NSE) and
TUNEL-positive motoneurons (TUNEL+ choline acetyltransferase,
ChAT) were double-stained in motor cortex, brain stem and spinal
cord – the regions that suffer the most from motoneuron degeneration
in ALS - and also in the sensory cortex in the three groups of mice, to
explore apoptosis in neurons and motoneurons in different regions
of the brain and spinal cord. Neuronal and glial apoptosis was
confirmed by their characteristic morphological features among the
three groups of mice via Transmission Electron Microscopy (TEM).
The DNA fragmentation and morphological characterization provide
sound evidence for whether SOD1 mutation induces motoneuron
apoptosis, whether apoptosis is specifically for motoneuron death,
and whether non-neuronal cells also suffer apoptosis in mSOD1
mice. Comparing the number of TUNEL-positive neurons between
motor and sensory cortices in the three groups of mice explores
whether in the mSOD1 mice motoneurons in the motor cortex are
more susceptible to apoptosis than are sensory neurons in the sensory
cortex. Together, these molecular and morphological results clarify
the equivocal and contradictory of previous reports regarding the
involvement of apoptosis in experimental ALS with strong in vivo
evidence, and exam whether apoptosis is a pathway in which non-
neuronal cells involved in motoneuron death in ALS.
MATERIALS AND METHODS
Animal use
The procedures using mice were approved by the University of
Texas Medical Branch Animal Care and Use Committee and were
in accord with the NIH Guide for the Care and Use of Laboratory
Animals. All efforts were made to use no more animals than needed
and to minimize their suffering by careful anesthesia.
ThreegroupsofmalemicepurchasedfromtheJacksonLaboratory
(Bar Harbor, ME, USA) were used: mSOD1 mice transfected with the
mutant (G93A) SOD1 gene from humans with fALS - B6SJL-TgN
(SOD1-G93A)1Gur; SOD1 mice transfected with the normal human
SOD1 gene - B6SJL-TgN (SOD1) 2Gur; and Nc mice - normal control
mice(B6SJLF1)withoutgenetransfection.Asreportedinourprevious
publication [38], we found no difference for our measurements when
using the littermates of mSOD1mice, the littermates of SOD1 mice
and the normal mice (B6SJLF1) as controls. Therefore, the B6SJLF1
normal control mice are a valid control for mSOD1 mice. Based on
our previous publications, 3 - 5 data from each group of mice were
sufficient for statistical analysis of significant difference [38,39]. In
the present study, 4 mSOD1, 4 SOD1, and 6 Nc mice were used for
double staining of TUNEL-positive neurons and motoneurons in
brain and spinal cord sections. Four additional mice for each group
were used for ELISA quantification of DNA fragmentation in brain
tissues. The mSOD1 mice died by 4 - 5 months; therefore, 3-month ±
1 week old mSOD1 mice and age-matched SOD1 and Nc mice were
used while paralysis was developing in the mutant strains. The mice
were delivered to our housing facility on different dates based on their
birthdays, so the surgical operation for tissues harvesting were also
performed on different dates to insure all tissues were obtained from
each mouse at the age of 3-month ± 1 week, so that the results are
comparable.
Tissue preparation and processing for staining
In all case the mouse was anesthetized with 3% halothane and
maintained with 0.5–1% halothane in an oxygen (3 L/min) and air
mixture (1:1) until the completion of surgery. After a craniotomy
and a laminectomy were performed at vertebrae T 10 - L5, the brain
and spinal cord were frozen in situ with liquid nitrogen, which
also sacrificed the animal. The tissue was then removed and stored
at -80°C. Before staining, the frozen brain and spinal cord were
fixed in 4% (w/v) paraformaldehyde in 0.01M phosphate-buffered
saline (PBS, pH 7.2-7.4) for 24 hours at 4o
C, then in 20% sucrose
in 0.01M PBS overnight at 4o
C. The brain was then cut coronally
at the bregma and frozen in liquid nitrogen. Coronal sections of 25
μm thickness were cut in rostral and caudal directions on a cryostat
(IMEB CUT 4500, Leica, Microsystems, Bensheim, Germany). The
spinal cords were processed similarly. All sections were collected and
kept in 15% sucrose and 0.1% sodium azide in PBS at 4o
C until use.
Sections selected from the frontal cortex (motor cortex), temporal
cortex (sensory cortex), brain stem (medulla oblongata containing
hypoglossal nerve nucleus), and spinal cord were processed by free-
floating staining as we previously described [39].
Characterization of apoptosis by DNA fragmentation
Apoptosis was characterized by assessing fragmented DNA as a
marker of apoptosis, and by morphological identification of specific
apoptotic features using TEM.
TUNEL-fluorescence staining of fragmented DNA: To
characterize specific DNA fragmentation, TUNEL staining was
performed using an ApopTag Fluorescein Direct in Situ Apoptosis
Detection Kit (Intergen Company, Purchase, NY, USA) following
the manufacture’s instruction. Briefly, the floating brain sections
were fixed in 1% paraformaldehyde in PBS for 1h and in pre-cooled
ethanol: acetic acid (2:1) for 5 min at –20°C. The fixed sections were
then immersed in equilibration buffer for 10 min, incubated with
TUNEL reagent in a humidified chamber at 37°C for 1 hour, and then
incubated in the stop/wash buffer at room temperature for 10 min to
stop the reaction. The floating sections were then mounted on slides,
coverslipped with PBS/glycerol and stored at 4°C. The fluorescence-
stained sections were observed under a Nikon epifluorescence
microscope with FITC optics (excitation: BP 450 - 490) to observe
TUNEL-positive cells.
TUNEL and immunohistochemical double staining of
fragmented DNA in neurons and motoneurons: After fixation and
quenching of the endogenous peroxidase in the floating sections
in 3.0% hydrogen peroxide in PBS for 5 min at room temperature,
TUNEL staining was performed using the ApopTag in Situ
Apoptosis Detection Kit (Intergen Company, Purchase, NY, USA)
and the reaction stopped as described above. The sections were then
washed with three changes of PBS for 1 min each and incubated in
anti-digoxigenin peroxidase conjugate for 30 min. After washing
in PBS, the sections were visualized with 0.05% diaminobenzidine
in staining buffer (0.05% M PBS, pH 7.6). Control sections were
treated similarly, but water or equilibration buffer was substituted
for the volume of TdT enzyme reagent. To identify TUNEL-positive
neurons, the TUNEL-stained brain sections were double-stained with
a monoclonal anti-NSE antibody (DAKO, Carpinteria, CA, USA) to
identify neurons in motor and sensory cortices. To identify TUNEL-
positive motoneurons, the TUNEL-stained sections in the spinal
cord and the hypoglossal nerve nucleus were double-stained with a
polyclonal antibody directed against ChAT (Abcam, Cambridge, UK).
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After TUNEL staining, the sections were washed in three changes of
PBS for 5 min each, blocked with 5% goat serum, and incubated with
an antibody to ChAT (1:200) or NSE (1:100). After washing in PBS,
the sections were incubated with biotinylated goat anti-rabbit-IgG
or goat anti-mouse-IgG (1:200, Sigma, St. Louis, MO, USA), then
with a solution of an avidin-biotin-horseradish peroxidase complex
(1:200, Vector Laboratories, Inc., Burlinghame, CA). The sections
were then visualized with a SG Substrate Kit (Vector Laboratories,
Inc., Burlinghame, CA), washed in water, dehydrated, cleaned and
coverslipped with Permount.
TEM Characterization of apoptosis by morphological
feature
Neuronal apoptosis was confirmed by its specific morphological
characteristics using our previously published TEM procedure
[40-42]. At 3 months, the mice were anesthetized and sacrificed
by transcardial perfusion-fixation with a solution consisting of 4%
paraformaldehyde and 2.0% glutaraldehyde in 0.1 M cacodylate
buffer (pH 7.2). The frontal cortices, brain stems, and spinal cords
(T1-T5) of the mice were removed following a craniotomy and a
laminectomy and kept in the same fixative overnight at 4°C. After
washing with cacodylate buffer, tissue blocks (0.1×0.1×0.1 mm) were
taken from the frontal cortex (layer III-V), brain stem (containing
the hypoglossal nerve nucleus) and spinal cord (ventral horn, Rexed
layer VIII) under an anatomical microscope and further fixed in 4%
osmium tetroxide in 0.1 M cacodylate buffer for 1 hour under a fume
hood. The blocks were stained overnight with 2% uranyl acetate at
4°C; the tissue blocks were then dehydrated and embedded in epoxy
resin. The semi-thin (1 μm) sections were stained with 1% toluidine
blue to locate apoptotic neurons; sections containing apoptotic
neurons were cut into ultra-thin sections with an ultramicrotome
(Reickert Ultra-cut S; Reichert, Depew, NY, USA), observed by TEM
at a magnification of X14,300-42,625 (Phillips CM-100; Philips,
Amsterdam, The Netherlands), and photographed.
Quantitative analysis of DNA fragmentation
DNA fragmentation in different brain regions was quantified
by counting the double-stained TUNEL-positive neurons. DNA
fragmentation in brain tissues was quantified by ELISA.
Quantification by counting TUNEL-positive neurons: For
quantitative evaluation of the differences between the motor cortex
and sensory cortex in terms of neuronal apoptosis, the TUNEL-
positive neurons in motor and sensory cortices were counted within
an area of 700 x 400 μm2
in layer II-VI of the cerebral cortex under a
microscope with a 10x field (Olympus BX40), or 40x for confirmation.
Three sequential sections in each animal were counted (3x each) and
the counts averaged.
ELISA quantification of DNA fragmentation: After a
craniotomy, the mouse brain was frozen in situ with liquid nitrogen
to avoid oxidative damage during cutting of the tissue; this also
sacrificed the animal. The brain was then removed and stored at
-80°C for analysis. The frozen brain was homogenized in lysis buffer
containing 8 M urea, 0.24 M sodium phosphate, 1% sodium dodecyl
sulfate and 10 mM EDTA, pH 6.8, using a 20:1 solution to tissue
ratio (v:w). The homogenates were extracted with an equal volume
of phenol-chloroform-isoamyl alcohol (25:24:1) saturated with the
homogenization buffer. The resulting emulsion was separated into
two phases by centrifugation at 4,500 g for 3 min. The aqueous phase
above the organic phase was removed for DNA analysis as reported
originally by Beland and coworkers [43] and slightly modified by us
[44]. Aliquots (10 μL) of each sample were used to quantitate the
nucleosome-associated apoptotic DNA fragments by ELISA using a
commercial cell death detection kit (Roche Boehringer Mannheim,
Indianapolis, IN, USA) that detects histone-associated DNA
fragments enriched in the cytoplasm of a dying cell. The analysis was
performed in duplicate according to the manufacturer’s instructions.
Statistical analysis
An unpaired t-test was used for comparing the difference in DNA
fragmentation between mSOD1 mice and each control measured by
ELISA in brain tissues. One-way ANOVA followed by the Turkey test
was used for statistical analysis of TUNEL-positive neurons in the
motor and sensory cortices of the three groups of mice. All data are
presented as mean ± SD.
RESULTS AND DISCUSSION
SOD1mutationsignificantlyincreasedDNAfragmentation
DNA fragmentation has frequently been used as an indicator of
apoptosis. In this study, DNA fragmentation was characterized as
a marker of apoptosis by fluorescence staining of TUNEL-positive
nuclei as described in the Materials and Methods. The upper panel
of figure 1 shows the typical TUNEL-fluorescence-stained nuclei
in the frontal cortices of an Nc mouse (A) and an mSOD1 mouse
(B) selected from the sections of 4 mSOD1 and 4 Nc mice. Very few
TUNEL-positive nuclei were seen in the control section (A). Many
TUNEL-positive nuclei were found in the section from mSOD1
mice (B), especially in layer V. This indicated that SOD1 mutation
increased the number of TUNEL-positive cells.
Figure 1: Characterization and quantification of DNA fragmentation: Upper
panel, photographs of the representive sections of TUNEL-fluorescence
stained mouse frontal cortices: (A) from an Nc mouse; (B) from an mSOD1
mouse. The SOD1 mutation apparently increased the number of TUNEL-
positive nuclei. Lower panel, ELISA quantification of DNA fragmentation in
brain tissues of three groups of mice. Significantly more DNA fragmentation
was found in mSOD1 mice than in SOD1 or Nc mice. Scale bar = 100 μm.
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The DNA fragmentation was also quantified by ELISA after
removal of the brain tissues from the three groups of mice as
described in the Materials and Methods (n = 4 for each group) and
shown in the lower panel of figure 1. The average absorbance (mean
± SD) was 1.10 ± 0.38 in normal control mice, 1.50 ± 0.55 in normal
SOD1-overexpressing mice, and 2.39 ± 0.35 in mutant SOD1 mice.
Unpaired t-tests demonstrated that there was significantly more DNA
fragmentation in mSOD1 mice than in SOD1 (p = 0.03) and Nc (p =
0.002) mice, with no significant difference between the two control
groups (p = 0.2). This result demonstrated that SOD1 mutation
induced apoptotic-like DNA fragmentation.
SOD1 mutation induced DNA fragmentation in
motoneurons
Since ALS is a motoneuron degenerative disorder, to determine
whether apoptosis is a pathway for motoneuron death in ALS,
motoneuron apoptosis was specifically assessed by double staining
with TUNEL and ChAT antibody in the brain stem and spinal
cord sections, as described in the Materials and Methods. The
motoneurons in the hypoglossal nerve nuclei of the brain stem
and the ventral horn of the spinal cord were observed from Nc,
SOD1 and mSOD1 mice (n = 4 for each group). Figure 2 shows
selected typical TUNEL + ChAT double stained sections. Almost
no apoptotic motoneurons were observed in the hypoglossal nerve
nuclei of the brain stem (Figure 2-left) and the ventral horn of the
spinal cord (Figure 2-right) from Nc (A and A’ in the left, and A - A”
in the right) and SOD1 (B and B’ in the left, and B - B” in the right)
mice. However, many TUNEL + ChAT-positive motoneurons were
observed in the sections from mSOD1 mice, both in the hypoglossal
nerve nuclei of the brain stem (C and C’ in Figure 2-left) and the
ventral gray matter of the spinal cords (C - C” in Figure 2-right). The
solid arrowheads in C’ (Figure 2-left) and C” (Figure 2-right) indicate
typical TUNEL-positive motoneurons. The fact that many TUNEL +
ChAT-positive motoneurons observed in the sections from mSOD1
mice in both regions, but almost no apoptotic motoneurons observed
in the corresponding areas in the SOD1 and Nc mice demonstrated
that SOD1 mutation induced apoptosis-like DNA fragmentation in
motoneurons.
It was noticed that many deeply stained TUNEL-positive nuclei
appeared in the sections from mSOD1 mice (C’ in the left and C” in
the right of Figure 2), as indicated by hollow arrowheads as examples.
This possibly represents apoptosis of other neurons and glial cells.
TEM confirmation of mSOD1 induced neuronal and glial
apoptosis
Since DNA fragmentation is not a specific marker for apoptosis;
the most reliable identification is via the morphological features of
apoptosis [33,37], so DNA fragmentation must be supported by
morphological observation. TEM is a gold-standard approach to
characterizeapoptosisbasedonspecificultra-structuralmorphological
changes such as cell surface blebbing, cell shrinkage, apoptotic
body formation, nuclear chromatin condensation, cytoplasmic
organelle compaction, and nuclear lobation [36,45]. In this study,
apoptotic neurons and glial cells were characterized by their specific
morphological features via TEM observation in ultra-thin sections of
the frontal cortex, brain stem (hypoglossal nerve nucleus) and spinal
cord ventral horn of the mSOD1 and Nc mice as shown in figure 3.
The neurons in the spinal cord and hypoglossal nerve nucleus of the
brain stem from normal mice were morphologically normal, showing
a large nucleus (N, pale-stained euchromatin) with abundant rough
endoplasmic reticulum (R, Figure 3A and D). The apoptotic neurons
in the spinal cord ventral horn (Figure 3B), hypoglossal nerve
nucleus (Figure 3E), and motor cortex (Figure 3H) from mSOD1
mice showed condensed chromatin accumulated in the nucleus and
condensed cytoplasm. Apoptotic glial cells were also observed in the
sections of the spinal cord (Figure 3C), hypoglossal nerve nucleus
(Figure 3F), and motor cortex (Figure 3, G and I) from the mSOD1
mice, characterized by condensed chromatin accumulated in the
Figure 2: DNA fragmentation in motoneurons: photographs of representive
double-stained sections of the brain stem (hypoglossal nerve nuclei, left
panel) and ventral horn of spinal cord (right panel). The motoneurons were
double-stained with TUNEL and an anti-ChAT antibody: (A) from Nc, (B) from
SOD1 and (C) from mSOD1 mice. A’, B’, and C’ are higher magnifications
of A, B and C, respectively. A”, B”, and C” in the right panels are higher
magnifications of A’, B’ and C’, respectively. Arrowheads in C’ (left panel)
and C” (right panel) of the mSOD1 mouse indicate typical TUNEL-positive
motoneurons. Arrows in A, B, and C indicate the central canal. Hollow
arrowheads in C’ and C” indicate TUNEL-positive non-motoneurons. Scale
bar = 100 μm.
Figure 3: TEM confirmation of neuronal and glial apoptosis: The tissues were
fixed and processed for TEM examination as described in the Materials and
Methods. A, B, and C from the spinal cord (magnification X10, 725, X24, 475,
and X18, 150, respectively) of Nc and mSOD1 mice as indicated. D, E, and
F from brain stem (hypoglossal nerve nucleus, magnification X10, 725, X14,
300 and X 24, 475, respectively) of Nc and mSOD1 mice, as indicated. G, H,
and I from the motor cortex of mSOD1 mice (magnification X 31, 625, X2 4,
475 and X 24, 475, respectively). Symbols: N, nucleus; R, rough endoplasmic
reticulum; Ch, chromatin; L, lipofuscin.
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nuclear rim. The apoptotic glial cells shown in Figure: 3C are from
the white matter of the spinal cord of an mSOD1 mouse, with one
cell in an early stage of apoptosis. Figure 3G from the motor cortex
of an mSOD1 mouse shows a normal neuron with a large nucleus
and abundant rough endoplasmic reticulum and an apoptotic
glial cell, characterized by condensed chromatin accumulated in
the nuclear rim. These TEM observations clearly show the typical
morphology of neuronal (Figure 3, B, E and H) and glial (Figure 3,
C, F, G, and I) apoptosis in all three of these regions in mSOD1 mice,
but no apoptotic cells were observed in Nc control mice (Figure 3,
A and D). Our findings of DNA fragmentation, together with our
structural observations by TEM, clearly demonstrated that 1) SOD1
mutation indeed induced apoptotic cell death in the motoneuron-
enriched regions; 2) apoptosis is one of the pathways causing cellular
degeneration and death in mSOD1 mice, and possibly in fALS; and
3) mSOD1-induced apoptosis is not specific to motoneurons, but is
also seen in other neurons and glial cells in the motoneuron-enriched
regions. Our finding that not only motoneurons but also glial cells
in the motoneuron-enriched regions die via apoptosis supports the
idea that neighboring non-motoneuron cells are involved in this
motoneuron degenerative disease, as mentioned in the Introduction.
Our observation of different stage of glial apoptosis in the white
matter of the spinal cord in mSOD1 mice (Figure 3C) indicates that
SOD1-induced glial apoptosis is wide spread, not limited to the gray
matter of the spinal cord.
It was noticed that a large lipofuscin granule (L) in the cytoplasm
appeared near the apoptotic glial cells from the motor cortex (Figure
3, G and I) and brain stem (Figure 3F) in mSOD1 mice. Number
of lipofuscin granules were also observed in the apoptotic neuron
(Figure 3E). The observation of large lipofuscin granules near the
apoptotic glial cells in all apoptotic glia-containing sections from the
grey matter of mSOD1 mice (Figure 3, F, G and I), and a number
of lipofuscin granules within an apoptotic neuron in a section of
an mSOD1 mouse (Figure 3E) suggests that lipofuscin might be
involved in the apoptotic cell death processes and play a role in the
apoptotic communications between glia and neurons in mSOD1
mice. Although no report can be found regarding this phenomenon,
whether the appearance of lipofuscin is related to the onset of cellular
apoptosis warrants further study.
SOD1mutationsignificantlyincreasedDNAfragmentation
in both the motor and sensory cortices
The present study compared neuronal DNA fragmentation
between the motor cortex and the sensory cortex. The frontal cortex
(motor cortex) and temporal cortex (sensory cortex) of the three
mouse groups (n = 4 per group) were double-stained with TUNEL
and an anti-NSE antibody as described in the Materials and Methods.
Figure 4 illustrates the typical double-stained sections of the motor
and sensory cortices selected from the sections in each mouse group.
Very few double-stained TUNEL-positive neurons were observed in
the sections from Nc and SOD1 mice in both the motor (left panel)
and sensory (right panel) cortices. Several double-stained TUNEL-
positive neurons were found in both cortices, especially in layer V
of the motor cortex and in layer II of the sensory cortex of mSOD1
mice as indicated by solid arrowheads in C”. TUNEL-positive nuclei
(Non-NSE positive cells) also appeared in both cortices in the sections
from mSOD1 mice (C” Figure 4). indicated by hollow arrowheads),
possibly representing glial cells. The unexpected finding that TUNEL-
positive neurons and possible glial cells not only appeared in the
motor cortex but also in the sensory cortex of mSOD1 mice suggested
that apoptosis are not specifically occur in the motoneuron-enriched
region and not only limited in the neurons in the later stage of disease.
To quantitatively compare the neuronal apoptosis between
motor and sensory regions, TUNEL-positive neurons were counted
in the sections from the three groups of mice (n = 4 per group). In
the motor cortex, the average number of TUNEL-positive neurons
was 0.75 ± 0.95 in Nc mice, 1.67 ± 1.86 in SOD1 mice, and 19.10
± 6.83 in mSOD1 mice. Statistical analysis by one-way ANOVA
followed by the Tukey test indicated that SOD1 mutation induced
significantly more TUNEL-positive neurons in the motor cortex of
mSOD1 mice than were seen in Nc mice (p < 0.001) or SOD1 mice
(p = 0.002), as shown in the top panel of figure 5. In the sensory
cortex, the average number of TUNEL-positive neurons was 0.42
± 0.50 in Nc mice, 0.78 ± 0.69 in SOD1 mice, and 12.30 ± 6.20 in
mSOD1 mice. The SOD1 mutation thus also significantly increased
Figure 4: DNA fragmentation in neurons of motor and sensory cortices:
Photographs of representive sections from the motor (left panel) and sensory
(right panel) cortices, double-stained with TUNEL and an anti-NSE antibody.
Section A from Nc, section B from SOD1, and section C from mSOD1 mice.
A’, A”, B’, B”, C’, and C” are higher magnifications of A, B and C, respectively.
Solid Arrowheads in C” indicate TUNEL-positive neurons. Hollow arrowheads
in C” indicate TUNEL-positive non-neuronal cells. Scale bar = 50 μm.
Figure 5: Quantitative comparison of DNA fragmentation between motor and
sensory cortices: TUNEL and anti-NSE antibody double-stained TUNEL-
positive neurons were counted in layers II-VI of the motor and sensory
cortices, as described in the Materials and Methods. The counts were
compared among the three groups of mice and statistically analyzed by
one-way ANOVA followed by the Tukey test. The number of TUNEL-positive
neurons was significantly higher in both the motor and sensory cortices of
mSOD1 mice than in Nc and SOD1 mice. There was no significant difference
between motor and sensory cortices in the mSOD1 mice.
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DNA fragmentation in the sensory cortex compared to Nc mice (p =
0.006) and to SOD1 mice (p = 0.01) (Figure 5, bottom panel). There
were no significant differences between Nc and SOD1 mice in either
the motor or sensory cortex. No significant difference was found for
the number of TUNEL-positive neurons in the motor cortex vs the
sensory cortex (p = 0.2) in the mSOD1 mice, although the average
number of apoptotic neurons in the motor cortex was higher than
in the sensory cortex (19.10 ± 6.83 vs. 12.30 ± 6.20). The finding
that SOD1 mutation induced significantly more TUNEL-positive
neurons in both the motor and sensory cortices compared to SOD1
and Nc mice (Figure 5) quantitatively demonstrated that apoptosis
is not limited to the motoneuron-enriched regions. This is contrary
to some earlier reports, such as apoptosis occur in the motor regions
(motor cortex and ventral horn of spinal cord) but not in the region
unaffected by ALS (somatosensory cortex) [34]; cortical interneuron
populations are apparently not affected in mSOD1 mice [13].
The mechanisms by which mSOD1 causes apoptosis are not
completely understood. It has been reported that oxidative stress and
mitochondrial dysfunction contribute to apoptosis in motoneurons of
mSOD1 Tg mice [9,12,34,46-50]. Using the G93A mSOD1 Tg models,
our previous studies demonstrate that mutation of SOD1 elevates the
in vivo levels of Reactive Species (RS), such as hydrogen peroxide
(H2
O2
), hydroxyl radical (.
OH), and nitric oxide. Consequently, the
oxidative and nitrative products malondialdehyde (an end product
of membrane lipid peroxidation), protein carbonyls (a marker for
protein oxidation), 8-hydroxy-2-deoxyguanosine (a marker of DNA
oxidation), and protein-bound nitrotyrosine are also significantly
elevated in mSOD1 mice compared to SOD1 and Nc mice [38,39].
Elevation of RS and resulting oxidative/nitrative damage in mSOD1
mice have also been reported by many other publications [51,52]
as examples. The demonstration of elevation of RS and all markers
of oxidation and nitration of protein, DNA and membrane lipids
in mSOD1 mice but not in SOD1 and Nc mice strongly supports
a correlation between SOD1 mutation and RS generation, with
consequent oxidative and nitrative damage. We also demonstrate
previously that the generation of .
OH, H2
O2
or peroxynitrite (ONOO-
) in an uninjured rat spinal cord induces oxidative and nitrative
damage [53,54] and necrotic and apoptotic cell death, confirmed
by TEM [40-42,55]. These findings demonstrate in vivo that RS
overproduction indeed induced apoptotic cell death. Together, these
results support a possible sequence of: SOD1 mutation → elevation
of RS → oxidative stress to major cellular components → apoptotic
cell death. We previously report that the number of neurons
positively immune-stained for all the markers of protein oxidation
and nitration is significantly higher in both the motor and sensory
cortices of mSOD1 mice than in controls [39]. The elevated oxidative
damage in both the motor and sensory cortices of mSOD1 mice may
cause apoptosis in both motor and sensory cortices of mSOD1 mice,
further supporting the causal relationship between oxidative stress
and apoptotic DNA fragmentation and explaining the reason that
apoptosis are not limited in the motoneuron-enriched brain regions.
Motoneuron apoptosis has been correlated with RS production and
oxidative stress in the mSOD1 model [14,56-58].
The SOD1 mutation, mitochondrial dysfunction, oxidative stress
and selective vulnerability of motoneurons are well reviewed by
Barber and Shaw [48]. Since SOD1 is ubiquitously expressed and not
restricted to motoneurons, it is understandable that motoneurons
are not the only cell type affected in ALS. However, motoneurons are
unusually large, with a cell body of approximately 50 – 60 μm and an
axon up to 1 m long in humans; therefore, SOD1 is present at higher
levels within motoneurons than in other types of cells [59]. The
higher level of SOD1 in motoneurons demands a high energy supply
from the cellular powerhouse - the mitochondria. In the case of SOD1
mutation, higher levels of mSOD1 and damaged mitochondria cause
increased RS production and thereby more severe oxidative stress,
and decreased mitochondrial efficiency for supplying energy in the
motoneurons of mSOD1 mice. This may lead more motoneurons
to go through RS and oxidative stress-induced apoptotic death.
Therefore the major consequence of SOD1 mutation is the selective
death of motoneuron populations, although other cells also suffer
apoptosis in ALS.
Insummary,usingmolecularandmorphologicalcharacterization,
the present study provides strong in vivo evidence that SOD1
mutation indeed induces motoneuron apoptosis in the motor cortex,
brain stem and ventral horn of the spinal cord; mSOD1-induced
apoptosis is not specific to motoneurons, but is also seen in other
neurons and glial cells in these motoneuron-enriched regions. This
suggests that apoptosis is not specifically for motoneuron death, but a
common pathway causing neuronal and glial degeneration and death
in mSOD1 mice, and possibly in fALS. It was also found that mSOD1
induces apoptosis in both motor cortex and sensory cortex and
there is no significant difference in apoptotic DNA fragmentation in
neurons between these regions. This indicates that apoptosis is not
specific to neuron death in motoneuron-enriched regions, but also
causes sensory neuron death in mSOD1 mice at the later stage of ALS.
Our TEM observation that large lipofuscin granules appeared near
the apoptotic glial cells in all apoptotic glia-containing sections from
the grey matter of mSOD1 mouse and a number of lipofuscin granules
were also found within an apoptotic neuron in a section of an mSOD1
mouse. Although the role of lipofuscin has not been reported, we
assume that lipofuscin might be involved in the apoptotic cell death
processes and play a role in the apoptotic communications between
glia and neurons in mSOD1 mice.
ACKNOWLEDGMENTS
This work was supported by a grant from the Amyotrophic
Lateral Sclerosis Association to D Liu and also supported by Dr. Liu’s
MSRDP account from the Department of Neurology, University
of Texas Medical Branch. The authors thank Dr. David Konkel for
editorial assistance during manuscript preparation.
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