Opposing effects of thyroid hormones on hypothalamic subunits and limbic structures in hyperthyroidism patients: A comprehensive volumetric study.

Thyroid hormones play a critical role in brain development, but paradoxically, patients with hyperthyroidism often exhibit cognitive decline and irritability. This study aims to explore the pattern of atrophy in hyperthyroid patients, changes in specific areas of the brain, including hypothalamic subfields and limbic structures, and their relationships with hormonal levels and psychometric tests. This prospective cross-sectional study involves 19 newly diagnosed, untreated hyperthyroid patients, and 15 age and gender-matched control subjects. The participants underwent psychometric and cognitive tests and volumetric MRI. The hypothalamic subfield (anterior-inferior, anterior-superior, superior-tubular, inferior-tubular, and posterior hypothalamus) and limbic structures (fornix, basal forebrain, nucleus accumbens, and septal nucleus) were segmented using voxel-based morphometry, surface-based morphometry, and deep learning algorithms. The groups were compared using the t-test, and correlation analyses were performed between clinical parameters and volumetric measurements. The correlation between hormonal parameters and volumetric measurements in patient and control groups was assessed with the Meng test. Hyperthyroid patients displayed widespread grey matter loss and sulcal shallowing in the left hemisphere. However, no local gyrification index changes or cortical thickness variations were detected. The limbic structures and hypothalamic subunits did not show any volume discrepancies. Free thyroxine in the patient group negatively correlated with bilateral anterior-inferior and right septal nucleus, but positively correlated with left anterior-inferior in the control group. Thyroid stimulating hormone in the patient group showed a positive correlation with bilateral fornix volume, a correlation absent in the control group. Disease duration negatively correlated with right anterior-inferior, right tubular inferior, and right septal nucleus. Changes in cognitive and psychometric test scores in the patient group correlated with the bilateral septal nucleus volume. Hyperthyroidism primarily leads to a reduction in grey matter volume and sulcal shallowing. Thyroid hormones have differing volumetric effects in limbic structures and hypothalamic subunits under physiological and hyperthyroid conditions.

Paediatric supratentorial tumours do not cause microstructural alterations in contralateral white matter: a preliminary study.

BACKGROUND AND PURPOSE: Intracranial tumours in children can exhibit different characteristics compared to those in adults. Understanding the microstructural changes in the contralateral normal-appearing white matter (NAWM) in children with primary intracranial masses is essential for optimizing treatment strategies. This study aimed to investigate the apparent diffusion coefficient (ADC) changes in contralateral NAWM using fully automated methods and deep learning algorithms. METHODS: We included 22 paediatric patients with primary supratentorial intracranial masses (23% high-grade) in the study. ADC values of the contralateral NAWM in the patient group were compared to those of a control group. Deep learning algorithms were utilized to analyse diffusion changes in NAWM. RESULTS: The mean ADC values of contralateral NAWM in the patient group were 0.80 ± 0.03 × 10-3 mm2/s, while the control group had a mean ADC value of 0.81 ± 0.03 × 10-3 mm2/s. There was no statistically significant difference between the groups (p = 0.39). Our findings indicate that there are no significant diffusion changes in the contralateral white matter of children with supratentorial intracranial masses. CONCLUSION: Primary supratentorial intracranial masses in children do not cause microstructural changes in contralateral normal-appearing white matter. This could be attributed to the less infiltrative nature and different biochemical profile of these tumour groups in the paediatric population. Further studies using advanced imaging techniques could provide additional insights into the distinct characteristics of paediatric intracranial tumours and improve patient management.

Microstructural Abnormalities in the Contralateral Normal-appearing White Matter of Glioblastoma Patients Evaluated with Advanced Diffusion Imaging.

PURPOSE: Glioblastoma patients develop recurrence in the opposite hemisphere far from the primary tumor site even after complete resection. This is one of the main reasons for short disease survival. Our aim in this study is to detect microstructural changes in the contralateral hemisphere of glioblastoma patients using different diffusion models with the fully automated tract-based spatial statistics (TBSS) method. METHODS: Fourteen right-sided and eleven left-sided glioblastoma patients without any treatment and eighteen age- and gender-matched controls were included in the study. Multi-shell diffusion weighted images were created with a 3T MRI device. After various preprocessing steps, images of fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD), axial kurtosis (AK), mean kurtosis (MK), radial kurtosis (RK), intracellular volume fraction (ICVF), orientation dispersion index (ODI), and isotropic water fraction (ISO) were obtained. TBSS was used to compare diffusion tensor imaging, diffusion kurtosis imaging, and neurite orientation dispersion and density imaging parameters of right- and left-sided glioblastoma patients with the control group for the contralateral hemisphere. RESULTS: Both right-sided and left-sided glioblastoma patients have shown an increase in MD and ODI in the contralateral hemisphere. While right-sided glioblastoma patients showed an increase in RD, AD, and ISO in a more limited area in the contralateral hemisphere, left-sided glioblastoma patients showed an increase in MK and AK. FA, ICVF, and RK did not show any difference in both groups. CONCLUSION: There are microstructural changes in the contralateral hemisphere in glioblastoma patients, and these changes differ between right-sided and left-sided glioblastoma patients.

Cortical morphological changes in multiple sclerosis patients: a study of cortical thickness, sulcal depth, and local gyrification index.

PURPOSE: Multiple sclerosis (MS) is a disease that progresses not only with demyelination but also with neurodegeneration. One of the goals of drug treatment in MS is to prevent neurodegeneration. Cortical thickness (CT), sulcal depth (SD), and local gyrification index (LGI) are indicators related to neurodegeneration. The aim of this study is to investigate changes in CT, SD, and LGI in patients with relapsing-remitting MS (RRMS). METHODS: T1 images of 74 RRMS patients and 65 healthy controls were used. T1 hypointense areas in RRMS patients were corrected using fully automated methods. CT, SD, and LGI were calculated for each patient. RESULTS: RRMS patients showed widespread cortical thinning, especially in bilateral temporoparietal areas, decreased SD in bilateral supramarginal gyrus, superior temporal gyrus, postcentral gyrus, and transverse temporal gyrus, and decreased LGI, especially in the left posterior cingulate gyrus and insula. The decrease in cortical thickness was associated with the number of attacks and lesion volume. EDSS was related to CT in the right lingual, inferior temporal, and fusiform gyrus. The LGI was correlated with T2 lesion volume in bilateral insula, with EDSS in the right insula and transverse and superior temporal gyri, and with the number of attacks in the right paracentral gyrus and pre-cuneus. However, SD did not show any correlation with EDSS, T2 lesion volume, or the number of attacks. CONCLUSION: Our results demonstrate widespread cortical thinning, decreased sulcal depth in local areas, and decreased gyrification in folds in RRMS patients, which are related to clinical parameters.

The role of MR diffusion kurtosis and neurite orientation dispersion and density imaging in evaluating gliomas.

BACKGROUND AND PURPOSE: Conventional MRI sequences in neuro-oncology are insufficient for glioma grading. However, newly developed diffusion-weighted imaging techniques have been shown to have a great potential for glioma grading. This study examined the diagnostic performance of diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), neurite orientation dispersion and density imaging (NODDI), and their combinations in glioma grading. METHODS: Multishell diffusion tensor images were obtained with 3T MRI in 38 glioma patients (22 high-grade glioma [HGG], 16 low-grade glioma [LGG]). DTI (fractional anisotropy [FA], mean diffusivity [MD], axial diffusivity [AD], radial diffusivity [RD]), DKI (Axial kurtosis [AK], mean kurtosis [MK], radial kurtosis [RK]), and NODDI (intracellular volume fraction [ICVF], orientation distribution index, isotropic water fraction [ISO]) images were obtained after preprocessing. The average value of these parameters was calculated in the solid components of the tumors. The receiver operating characteristic curve analyses were performed to investigate the diagnostic performance and the curves were compared with the Delong test. RESULTS: FA shows an increase in HGG, while MD, RD, and AD exhibit a decrease. AK, MK, and RK were higher in HGG than LGG. ICVF increased in HGG, while ISO decreased. AK demonstrated the best diagnostic performance among all parameters, and kurtosis outperformed NODDI but not DTI. Combining these parameters did not yield a statistically significant improvement in diagnostic performance. CONCLUSION: DTI, DKI, and NODDI approaches can differentiate between HGG and LGG; however, kurtosis parameters perform better and adding NODDI parameters does not improve diagnostic performance. Using multishell b-value has not led to an increase in diagnostic performance.

Novel rAAV vector mediated intrathecal HGF delivery has an impact on neuroimmune modulation in the ALS motor cortex with TDP-43 pathology.

Recombinant adeno-associated virus (rAAV)-based gene therapies offer an immense opportunity for rare diseases, such as amyotrophic lateral sclerosis (ALS), which is defined by the loss of the upper and the lower motor neurons. Here, we describe generation, characterization, and utilization of a novel vector system, which enables expression of the active form of hepatocyte growth factor (HGF) under EF-1α promoter with bovine growth hormone (bGH) poly(A) sequence and is effective with intrathecal injections. HGF's role in promoting motor neuron survival had been vastly reported. Therefore, we investigated whether intrathecal delivery of HGF would have an impact on one of the most common pathologies of ALS: the TDP-43 pathology. Increased astrogliosis, microgliosis and progressive upper motor neuron loss are important consequences of ALS in the motor cortex with TDP-43 pathology. We find that cortex can be modulated via intrathecal injection, and that expression of HGF reduces astrogliosis, microgliosis in the motor cortex, and help restore ongoing UMN degeneration. Our findings not only introduce a novel viral vector for the treatment of ALS, but also demonstrate modulation of motor cortex by intrathecal viral delivery, and that HGF treatment is effective in reducing astrogliosis and microgliosis in the motor cortex of ALS with TDP-43 pathology.

SBT-272 improves TDP-43 pathology in ALS upper motor neurons by modulating mitochondrial integrity, motility, and function.

Mitochondrial defects are one of the common underlying causes of neuronal vulnerability in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), and TDP-43 pathology is the most commonly observed proteinopathy. Disrupted inner mitochondrial membrane (IMM) reported in the upper motor neurons (UMNs) of ALS patients with TDP-43 pathology is recapitulated in the UMNs of well-characterized hTDP-43 mouse model of ALS. The construct validity, such as shared and common cellular pathology in mice and human, offers a unique opportunity to test treatment strategies that may translate to patients. SBT-272 is a well-tolerated brain-penetrant small molecule that stabilizes cardiolipin, a phospholipid found in IMM, thereby restoring mitochondrial structure and respiratory function. We investigated whether SBT-272 can improve IMM structure and health in UMNs diseased with TDP-43 pathology in our well-characterized UMN reporter line for ALS. We found that SBT-272 significantly improved mitochondrial structural integrity and restored mitochondrial motility and function. This led to improved health of diseased UMNs in vitro. In comparison to edaravone and AMX0035, SBT-272 appeared more effective in restoring health of diseased UMNs. Chronic treatment of SBT-272 for sixty days starting at an early symptomatic stage of the disease in vivo led to a significant reduction in astrogliosis, microgliosis, and TDP-43 pathology in the ALS motor cortex. Our results underscore the therapeutic potential of SBT-272, especially within the context of TDP-43 pathology and mitochondrial dysfunction.

Volumetric changes in hypothalamic subunits in patients with relapsing remitting multiple sclerosis.

PURPOSE: Studies on hypothalamic changes in patients with relapsing remitting multiple sclerosis (RRMS) are very scarce, despite the fact that the relationship with the hypothalamus is frequently reported. The aim of the study was to determine the volume of the hypothalamic subunits and the total hypothalamus and its relationship with the total demyelinating lesion volume (TLV) and expanded disability status scale (EDSS) in RRMS patients. METHODS: In this cross-sectional study, anterior-superior, superior tubular, posterior hypothalamus, anterior-inferior, inferior tubular subunits of hypothalamus, and total hypothalamus volume were calculated, with fully automatic analysis methods using volumetric T1 images of 65 relapsed RRMS patients and 68 healthy controls (HC). Volume changes in the hypothalamus and its subunits in RRMS patients were examined using multivariate analysis of covariance (MANCOVA). The relationship of these volumes with EDSS and TLV was investigated by partial correlation analysis. RESULTS: There is volume reduction in total hypothalamus (F = 13.87, p < 0.001), anterior-superior (F = 19.2, p < 0.001), superior tubular (F = 10.1, p = 0.002) subunits, and posterior hypothalamus (F = 19.2, p < 0.001) volume in RRMS patients. EDSS correlates negatively with anterior-superior (p = 0.017, r = - 0.333), superior tubular subunits (p = 0.023, r = - 0.439), posterior hypothalamus (p < 0.001, r = - 0.511), and whole hypothalamus volume (p = 0.001, r = - 0.439). TLV correlates negatively with anterior superior (p < 0.001, r = - 0.565), anterior inferior (p = 0.002, r = - 0.431), superior tubular subunits (p = 0.002, r = - 0.432), posterior hypothalamus (p < 0.001, r = - 0.703), and whole hypothalamus (p < 0.001, r = - 0.627) volumes. CONCLUSION: This study demonstrates a reduction in total hypothalamus volume, anterior-superior, superior tubular, and posterior hypothalamus in patients with RRMS. Anterior-superior and superior tubular subunit, posterior hypothalamus, and total hypothalamus volume were negatively correlated with TLV and EDSS scores.

Quantitative evaluation of cortical thickness in 3T in Behçet's patients without neurological involvement and parenchymal neuro-Behçet's disease.

PURPOSE: To evaluate the spatial distribution of cortical damage in Behcet's Disease (BD) with or without neurological involvement using a cortical thickness measurement approach using three-dimensional T1-weighted imaging. MATERIAL AND METHODS: Fifty-eight BD patients without neurological involvement, twenty-two Parenchymal Neuro-Behçets disease (PNBD) patients, and fifty healthy controls were included in the prospective study. Anatomical 3D T1 images were obtained from all participants using 3T MRI. Using a computational anatomy toolbox (CAT12), we calculated and compared group differences in cortical thickness. RESULTS: Patients with BD without neurological involvement showed cortical thickness reduction in bilateral frontal, bilateral parietal, and right precuneus compared with the healthy controls (HCs) (p < 0.05 FWE corrected [FWEc]). PNBD patients showed frontoparietal cortical thickness reduction when compared with BD patients without neurological involvement (p < 0.05 FWEc). The PNBD patients showed widespread cortical thickness reduction compared with the HC patients (p < 0.05 FWEc). Disease duration was correlated with cortical thickness in the right pericalcarine (p = 0.012 false discovery rate [FDR], r = -0.40), left pericalcarine (p = 0.013 FDR, r = -0.44), and left transverse temporal (p = 0.007 FDR, r = -0.41) regions. CONCLUSION: There is a decrease in cortical thickness in BD patients without neurological involvement. Cortical thickness reduction is more prominent in parenchymal neurobehçet's patients. Cortical thickness shows a negative correlation with disease duration in some regions.

NU-9 improves health of hSOD1G93A mouse upper motor neurons in vitro, especially in combination with riluzole or edaravone.

Even though amyotrophic lateral sclerosis (ALS) is a disease of the upper and lower motor neurons, to date none of the compounds in clinical trials have been tested for improving the health of diseased upper motor neurons (UMNs). There is an urgent need to develop preclinical assays that include UMN health as a readout. Since ALS is a complex disease, combinatorial treatment strategies will be required to address the mechanisms perturbed in patients. Here, we describe a novel in vitro platform that takes advantage of an UMN reporter line in which UMNs are genetically labeled with fluorescence and have misfolded SOD1 toxicity. We report that NU-9, an analog of the cyclohexane-1,3-dione family of compounds, improves the health of UMNs with misfolded SOD1 toxicity more effectively than riluzole or edaravone, -the only two FDA-approved ALS drugs to date-. Interestingly, when NU-9 is applied in combination with riluzole or edaravone, there is an additive effect on UMN health, as they extend longer axons and display enhanced branching and arborization, two important characteristics of healthy UMNs in vitro.

Upper motor neurons are a target for gene therapy and UCHL1 is necessary and sufficient to improve cellular integrity of diseased upper motor neurons.

There are no effective cures for upper motor neuron (UMN) diseases, such as amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, and hereditary spastic paraplegia. Here, we show UMN loss occurs independent of spinal motor neuron degeneration and that UMNs are indeed effective cellular targets for gene therapy, which offers a potential solution especially for UMN disease patients. UCHL1 (ubiquitin C-terminal hydrolase-L1) is a deubiquitinating enzyme crucial for maintaining free ubiquitin levels. Corticospinal motor neurons (CSMN, a.k.a UMNs in mice) show early, selective, and profound degeneration in Uchl1nm3419 (UCHL1-/-) mice, which lack all UCHL1 function. When UCHL1 activity is ablated only from spinal motor neurons, CSMN remained intact. However, restoring UCHL1 specifically in CSMN of UCHL1-/- mice via directed gene delivery was sufficient to improve CSMN integrity to the healthy control levels. In addition, when UCHL1 gene was delivered selectively to CSMN that are diseased due to misfolded SOD1 toxicity and TDP-43 pathology via AAV-mediated retrograde transduction, the disease causing misfolded SOD1 and mutant human TDP-43 were reduced in hSOD1G93A and prpTDP-43A315T models, respectively. Diseased CSMN retained their neuronal integrity and cytoarchitectural stability in two different mouse models that represent two distinct causes of neurodegeneration in ALS.

Improving mitochondria and ER stability helps eliminate upper motor neuron degeneration that occurs due to mSOD1 toxicity and TDP-43 pathology.

BACKGROUND: Upper motor neurons (UMNs) are a key component of motor neuron circuitry. Their degeneration is a hallmark for diseases, such as hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), and amyotrophic lateral sclerosis (ALS). Currently there are no preclinical assays investigating cellular responses of UMNs to compound treatment, even for diseases of the UMNs. The basis of UMN vulnerability is not fully understood, and no compound has yet been identified to improve the health of diseased UMNs: two major roadblocks for building effective treatment strategies. METHODS: Novel UMN reporter models, in which UMNs that are diseased because of misfolded superoxide dismutase protein (mSOD1) toxicity and TDP-43 pathology are labeled with eGFP expression, allow direct assessment of UMN response to compound treatment. Electron microscopy reveals very precise aspects of endoplasmic reticulum (ER) and mitochondrial damage. Administration of NU-9, a compound initially identified based on its ability to reduce mSOD1 toxicity, has profound impact on improving the health and stability of UMNs, as identified by detailed cellular and ultrastructural analyses. RESULTS: Problems with mitochondria and ER are conserved in diseased UMNs among different species. NU-9 has drug-like pharmacokinetic properties. It lacks toxicity and crosses the blood brain barrier. NU-9 improves the structural integrity of mitochondria and ER, reduces levels of mSOD1, stabilizes degenerating UMN apical dendrites, improves motor behavior measured by the hanging wire test, and eliminates ongoing degeneration of UMNs that become diseased both because of mSOD1 toxicity and TDP-43 pathology, two distinct and important overarching causes of motor neuron degeneration. CONCLUSIONS: Mechanism-focused and cell-based drug discovery approaches not only addressed key cellular defects responsible for UMN loss, but also identified NU-9, the first compound to improve the health of diseased UMNs, neurons that degenerate in ALS, HSP, PLS, and ALS/FTLD patients.

The Timing and Extent of Motor Neuron Vulnerability in ALS Correlates with Accumulation of Misfolded SOD1 Protein in the Cortex and in the Spinal Cord.

Understanding the cellular and molecular basis of selective vulnerability has been challenging, especially for motor neuron diseases. Developing drugs that improve the health of neurons that display selective vulnerability relies on in vivo cell-based models and quantitative readout measures that translate to patient outcome. We initially developed and characterized UCHL1-eGFP mice, in which motor neurons are labeled with eGFP that is stable and long-lasting. By crossing UCHL1-eGFP to amyotrophic lateral sclerosis (ALS) disease models, we generated ALS mouse models with fluorescently labeled motor neurons. Their examination over time began to reveal the cellular basis of selective vulnerability even within the related motor neuron pools. Accumulation of misfolded SOD1 protein both in the corticospinal and spinal motor neurons over time correlated with the timing and extent of degeneration. This further proved simultaneous degeneration of both upper and lower motor neurons, and the requirement to consider both upper and lower motor neuron populations in drug discovery efforts. Demonstration of the direct correlation between misfolded SOD1 accumulation and motor neuron degeneration in both cortex and spinal cord is important for building cell-based assays in vivo. Our report sets the stage for shifting focus from mice to diseased neurons for drug discovery efforts, especially for motor neuron diseases.

Complexity of Generating Mouse Models to Study the Upper Motor Neurons: Let Us Shift Focus from Mice to Neurons.

Motor neuron circuitry is one of the most elaborate circuitries in our body, which ensures voluntary and skilled movement that requires cognitive input. Therefore, both the cortex and the spinal cord are involved. The cortex has special importance for motor neuron diseases, in which initiation and modulation of voluntary movement is affected. Amyotrophic lateral sclerosis (ALS) is defined by the progressive degeneration of both the upper and lower motor neurons, whereas hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS) are characterized mainly by the loss of upper motor neurons. In an effort to reveal the cellular and molecular basis of neuronal degeneration, numerous model systems are generated, and mouse models are no exception. However, there are many different levels of complexities that need to be considered when developing mouse models. Here, we focus our attention to the upper motor neurons, which are one of the most challenging neuron populations to study. Since mice and human differ greatly at a species level, but the cells/neurons in mice and human share many common aspects of cell biology, we offer a solution by focusing our attention to the affected neurons to reveal the complexities of diseases at a cellular level and to improve translational efforts.

Evidence for an early innate immune response in the motor cortex of ALS.

BACKGROUND: Recent evidence indicates the importance of innate immunity and neuroinflammation with microgliosis in amyotrophic lateral sclerosis (ALS) pathology. The MCP1 (monocyte chemoattractant protein-1) and CCR2 (CC chemokine receptor 2) signaling system has been strongly associated with the innate immune responses observed in ALS patients, but the motor cortex has not been studied in detail. METHODS: After revealing the presence of MCP1 and CCR2 in the motor cortex of ALS patients, to elucidate, visualize, and define the timing, location and the extent of immune response in relation to upper motor neuron vulnerability and progressive degeneration in ALS, we developed MCP1-CCR2-hSOD1G93A mice, an ALS reporter line, in which cells expressing MCP1 and CCR2 are genetically labeled by monomeric red fluorescent protein-1 and enhanced green fluorescent protein, respectively. RESULTS: In the motor cortex of MCP1-CCR2-hSOD1G93A mice, unlike in the spinal cord, there was an early increase in the numbers of MCP1+ cells, which displayed microglial morphology and selectively expressed microglia markers. Even though fewer CCR2+ cells were present throughout the motor cortex, they were mainly infiltrating monocytes. Interestingly, MCP1+ cells were found in close proximity to the apical dendrites and cell bodies of corticospinal motor neurons (CSMN), further implicating the importance of their cellular interaction to neuronal pathology. Similar findings were observed in the motor cortex of ALS patients, where MCP1+ microglia were especially in close proximity to the degenerating apical dendrites of Betz cells. CONCLUSIONS: Our findings reveal that the intricate cellular interplay between immune cells and upper motor neurons observed in the motor cortex of ALS mice is indeed recapitulated in ALS patients. We generated and characterized a novel model system, to study the cellular and molecular basis of this close cellular interaction and how that relates to motor neuron vulnerability and progressive degeneration in ALS.

Apical dendrite degeneration, a novel cellular pathology for Betz cells in ALS.

Apical dendrites of Betz cells are important sites for the integration of cortical input, however their health has not been fully assessed in ALS patients. We investigated the primary motor cortices isolated from post-mortem normal control subjects, patients with familial ALS (fALS), sporadic ALS (sALS), ALS with frontotemporal dementia (FTD-ALS), and Alzheimer's disease (AD), and found profound apical dendrite degeneration of Betz cells in both fALS and sALS, as well as FTD-ALS patients. In contrast, Betz cells of AD patients and normal controls retain cellular integrity in the motor cortex, and CA1 pyramidal neurons show abnormalities predominantly within their soma, rather than the apical dendrite. In line with extensive vacuolation and cytoarchitectural disintegration, the numbers of synapses were also significantly reduced only in ALS patients. Our findings indicate apical dendrite degeneration as a novel cellular pathology that distinguishes ALS and further support the importance of cortical dysfunction for disease pathology.

Complementary feature selection from alternative splicing events and gene expression for phenotype prediction.

MOTIVATION: A central task of bioinformatics is to develop sensitive and specific means of providing medical prognoses from biomarker patterns. Common methods to predict phenotypes in RNA-Seq datasets utilize machine learning algorithms trained via gene expression. Isoforms, however, generated from alternative splicing, may provide a novel and complementary set of transcripts for phenotype prediction. In contrast to gene expression, the number of isoforms increases significantly due to numerous alternative splicing patterns, resulting in a prioritization problem for many machine learning algorithms. This study identifies the empirically optimal methods of transcript quantification, feature engineering and filtering steps using phenotype prediction accuracy as a metric. At the same time, the complementary nature of gene and isoform data is analyzed and the feasibility of identifying isoforms as biomarker candidates is examined. RESULTS: Isoform features are complementary to gene features, providing non-redundant information and enhanced predictive power when prioritized and filtered. A univariate filtering algorithm, which selects up to the N highest ranking features for phenotype prediction is described and evaluated in this study. An empirical comparison of pipelines for isoform quantification is reported by performing cross-validation prediction tests with datasets from human non-small cell lung cancer (NSCLC) patients, human patients with chronic obstructive pulmonary disease (COPD) and amyotrophic lateral sclerosis (ALS) transgenic mice, each including samples of diseased and non-diseased phenotypes. AVAILABILITY AND IMPLEMENTATION: https://github.com/clabuzze/Phenotype-Prediction-Pipeline.git CONTACT: clabuzze@iastate.edu, antoniom@bc.edu, watsondk@musc.edu, andersonpe2@cofc.edu.

Absence of UCHL 1 function leads to selective motor neuropathy.

OBJECTIVE: The aim of this study was to investigate the role of ubiquitin C-terminal hydrolase-L1 (UCHL1) for motor neuron circuitry and especially in spinal motor neuron (SMN) health, function, and connectivity. METHODS: Since mutations in UCHL1 gene leads to motor dysfunction in patients, we investigated the role of UCHL1 on SMN survival, axon health, and connectivity with the muscle, by employing molecular and cellular marker expression analysis and electrophysiological recordings, in healthy wild-type and Uchl1 (nm3419) (UCHL1-/-) mice, which lack all UCHL1 function. RESULTS: There is pure motor neuropathy with selective degeneration of the motor, but not sensory axons in the absence of UCHL1 function. Neuromuscular junctions (NMJ) are impaired in muscle groups that are innervated by slow-twitch or fast-twitch SMN. However, unlike corticospinal motor neurons, SMN cell bodies remain intact with no signs of elevated endoplasmic reticulum (ER) stress. INTERPRETATION: Presence of NMJ defects and progressive retrograde axonal degeneration in the absence of major SMN soma loss suggest that defining pathology as a function of neuron number is misleading and that upper and lower motor neurons utilize UCHL1 function in different cellular events. In line with findings in patients with mutations in UCHL1 gene, our results suggest a unique role of UCHL1, especially for motor neuron circuitry. SMN require UCHL1 to maintain NMJ and motor axon health, and that observed motor dysfunction in the absence of UCHL1 is not due to SMN loss, but mostly due to disintegrated circuitry.

Visualization of Sensory Neurons and Their Projections in an Upper Motor Neuron Reporter Line.

Visualization of peripheral nervous system axons and cell bodies is important to understand their development, target recognition, and integration into complex circuitries. Numerous studies have used protein gene product (PGP) 9.5 [a.k.a. ubiquitin carboxy-terminal hydrolase L1 (UCHL1)] expression as a marker to label sensory neurons and their axons. Enhanced green fluorescent protein (eGFP) expression, under the control of UCHL1 promoter, is stable and long lasting in the UCHL1-eGFP reporter line. In addition to the genetic labeling of corticospinal motor neurons in the motor cortex and degeneration-resistant spinal motor neurons in the spinal cord, here we report that neurons of the peripheral nervous system are also fluorescently labeled in the UCHL1-eGFP reporter line. eGFP expression is turned on at embryonic ages and lasts through adulthood, allowing detailed studies of cell bodies, axons and target innervation patterns of all sensory neurons in vivo. In addition, visualization of both the sensory and the motor neurons in the same animal offers many advantages. In this report, we used UCHL1-eGFP reporter line in two different disease paradigms: diabetes and motor neuron disease. eGFP expression in sensory axons helped determine changes in epidermal nerve fiber density in a high-fat diet induced diabetes model. Our findings corroborate previous studies, and suggest that more than five months is required for significant skin denervation. Crossing UCHL1-eGFP with hSOD1G93A mice generated hSOD1G93A-UeGFP reporter line of amyotrophic lateral sclerosis, and revealed sensory nervous system defects, especially towards disease end-stage. Our studies not only emphasize the complexity of the disease in ALS, but also reveal that UCHL1-eGFP reporter line would be a valuable tool to visualize and study various aspects of sensory nervous system development and degeneration in the context of numerous diseases.

Corticospinal Motor Neurons Are Susceptible to Increased ER Stress and Display Profound Degeneration in the Absence of UCHL1 Function.

Corticospinal motor neurons (CSMN) receive, integrate, and relay cerebral cortex's input toward spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases. Previously, 5 patients with mutations in the ubiquitin carboxy-terminal hydrolase-L1 (UCHL1) gene were reported to have neurodegeneration and motor neuron dysfunction with upper motor neuron involvement. To investigate the role of UCHL1 on CSMN health and stability, we used both in vivo and in vitro approaches, and took advantage of the Uchl1(nm3419) (UCHL1(-/-)) mice, which lack all UCHL1 function. We report a unique role of UCHL1 in maintaining CSMN viability and cellular integrity. CSMN show early, selective, progressive, and profound cell loss in the absence of UCHL1. CSMN degeneration, evident even at pre-symptomatic stages by disintegration of the apical dendrite and spine loss, is mediated via increased ER stress. These findings bring a novel understanding to the basis of CSMN vulnerability, and suggest UCHL1(-/-) mice as a tool to study CSMN pathology.