Patterns of CAG repeat instability in the central nervous system and periphery in Huntington's disease and in spinocerebellar ataxia type 1.

The expanded HTT CAG repeat causing Huntington's disease (HD) exhibits somatic expansion proposed to drive the rate of disease onset by eliciting a pathological process that ultimately claims vulnerable cells. To gain insight into somatic expansion in humans, we performed comprehensive quantitative analyses of CAG expansion in ~50 central nervous system (CNS) and peripheral postmortem tissues from seven adult-onset and one juvenile-onset HD individual. We also assessed ATXN1 CAG repeat expansion in brain regions of an individual with a neurologically and pathologically distinct repeat expansion disorder, spinocerebellar ataxia type 1 (SCA1). Our findings reveal similar profiles of tissue instability in all HD individuals, which, notably, were also apparent in the SCA1 individual. CAG expansion was observed in all tissues, but to different degrees, with multiple cortical regions and neostriatum tending to have the greatest instability in the CNS, and liver in the periphery. These patterns indicate different propensities for CAG expansion contributed by disease locus-independent trans-factors and demonstrate that expansion per se is not sufficient to cause cell type or disease-specific pathology. Rather, pathology may reflect distinct toxic processes triggered by different repeat lengths across cell types and diseases. We also find that the HTT CAG length-dependent expansion propensity of an individual is reflected in all tissues and in cerebrospinal fluid. Our data indicate that peripheral cells may be a useful source to measure CAG expansion in biomarker assays for therapeutic efforts, prompting efforts to dissect underlying mechanisms of expansion that may differ between the brain and periphery.

SCA8 RAN polySer protein preferentially accumulates in white matter regions and is regulated by eIF3F.

Spinocerebellar ataxia type 8 (SCA8) is caused by a bidirectionally transcribed CTG·CAG expansion that results in the in vivo accumulation of CUG RNA foci, an ATG-initiated polyGln and a polyAla protein expressed by repeat-associated non-ATG (RAN) translation. Although RAN proteins have been reported in a growing number of diseases, the mechanisms and role of RAN translation in disease are poorly understood. We report a novel toxic SCA8 polySer protein which accumulates in white matter (WM) regions as aggregates that increase with age and disease severity. WM regions with polySer aggregates show demyelination and axonal degeneration in SCA8 human and mouse brains. Additionally, knockdown of the eukaryotic translation initiation factor eIF3F in cells reduces steady-state levels of SCA8 polySer and other RAN proteins. Taken together, these data show polySer and WM abnormalities contribute to SCA8 and identify eIF3F as a novel modulator of RAN protein accumulation.

Polyglutamine spinocerebellar ataxias - from genes to potential treatments.

The dominantly inherited spinocerebellar ataxias (SCAs) are a large and diverse group of neurodegenerative diseases. The most prevalent SCAs (SCA1, SCA2, SCA3, SCA6 and SCA7) are caused by expansion of a glutamine-encoding CAG repeat in the affected gene. These SCAs represent a substantial portion of the polyglutamine neurodegenerative disorders and provide insight into this class of diseases as a whole. Recent years have seen considerable progress in deciphering the clinical, pathological, physiological and molecular aspects of the polyglutamine SCAs, with these advances establishing a solid base from which to pursue potential therapeutic approaches.

Polarization-sensitive optical coherence tomography reveals gray matter and white matter atrophy in SCA1 mouse models.

Spinocerebellar ataxia type 1 (SCA1) is a fatal inherited neurodegenerative disease. In this study, we demonstrate the label-free optical imaging methodology that can detect, with a high degree of sensitivity, discrete areas of degeneration in the cerebellum of the SCA1 mouse models. We used ATXN1[82Q] and ATXN1[30Q]-D776 mice in which the transgene is directed only to Purkinje cells. Molecular layer, granular layer, and white matter regions are analyzed using the intrinsic contrasts provided by polarization-sensitive optical coherence tomography. Cerebellar atrophy in SCA1 mice occurred both in gray matter and white matter. While gray matter atrophy is obvious, indications of white matter atrophy including different birefringence characteristics, and shortened and contorted branches are observed. Imaging results clearly show the loss or atrophy of myelinated axons in ATXN1[82Q] mice. The method provides unbiased contrasts that can facilitate the understanding of the pathological progression in neurodegenerative diseases and other neural disorders.

Assessing recovery from neurodegeneration in spinocerebellar ataxia 1: Comparison of in vivo magnetic resonance spectroscopy with motor testing, gene expression and histology.

Suppression of transgene expression in a conditional transgenic mouse model of spinocerebellar ataxia 1 (SCA1) reverses the Purkinje cell pathology and motor dysfunction that are hallmarks of SCA1. We previously showed that cerebellar neurochemical levels measured by magnetic resonance spectroscopy (MRS) correlate with progression of pathology and clinical status of patients and that abnormal neurochemical levels normalize upon suppression of transgene expression, indicating their potential as robust surrogate markers of treatment effects. Here we investigated the relative sensitivities of MRS, histology, transgene expression and motor behavioral testing to disease reversal in conditional SCA1 mice. Transgene expression was suppressed by doxycycline administration and treated and untreated mice were assessed by MRS at 9.4tesla before and after treatment and with an accelerating Rotarod, histology and quantitative polymerase chain reaction (qPCR) for ataxin-1 transgene expression following doxycycline treatment. The MRS-measured N-acetylaspartate-to-myo-inositol ratio (NAA/Ins) correlated significantly with the molecular layer (ML) thickness and transgene expression. NAA/Ins, ML thickness and transgene expression were highly significantly different between the treated vs. untreated groups (p<0.0001), while the Rotarod assessment showed a trend for treatment effect. MRS, qPCR and histology had high sensitivity/specificity to distinguish treated from untreated mice, all with areas under the curve (AUC)=0.97-0.98 in receiver operating characteristic (ROC) analyses, while Rotarod had significantly lower sensitivity and specificity (AUC=0.72). Therefore, MRS accurately reflects the extent of recovery from neurodegeneration with sensitivity similar to invasive measures, further validating its potential as a surrogate marker in pre-clinical and clinical treatment trials.

Multiple calcifying pseudoneoplasms of the neuraxis.

Calcified pseudoneoplasms of the neuraxis (CAPNON) are a rare diagnostic entity. They have been reported intra-cranially as well as in the spine, and are most commonly found at the skull base. This is a case report of a 38-year-old woman who presented with bilateral CAPNON, diagnosed 8 years apart. While there are approximately 46 reported in the current literature of cerebral and spinal fibro-osseous lesions, this is the first report of separate lesions.

Bidirectional expression of CUG and CAG expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8.

We previously reported that a (CTG)n expansion causes spinocerebellar ataxia type 8 (SCA8), a slowly progressive ataxia with reduced penetrance. We now report a transgenic mouse model in which the full-length human SCA8 mutation is transcribed using its endogenous promoter. (CTG)116 expansion, but not (CTG)11 control lines, develop a progressive neurological phenotype with in vivo imaging showing reduced cerebellar-cortical inhibition. 1C2-positive intranuclear inclusions in cerebellar Purkinje and brainstem neurons in SCA8 expansion mice and human SCA8 autopsy tissue result from translation of a polyglutamine protein, encoded on a previously unidentified antiparallel transcript (ataxin 8, ATXN8) spanning the repeat in the CAG direction. The neurological phenotype in SCA8 BAC expansion but not BAC control lines demonstrates the pathogenicity of the (CTG-CAG)n expansion. Moreover, the expression of noncoding (CUG)n expansion transcripts (ataxin 8 opposite strand, ATXN8OS) and the discovery of intranuclear polyglutamine inclusions suggests SCA8 pathogenesis involves toxic gain-of-function mechanisms at both the protein and RNA levels.

Spectrin mutations cause spinocerebellar ataxia type 5.

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

Purkinje cell ataxin-1 modulates climbing fiber synaptic input in developing and adult mouse cerebellum.

Previous studies indicate that while transgenic mice with ATXN1[30Q]-D776-induced disease share pathological features caused by ATXN1[82Q] having an expanded polyglutamine tract, they fail to manifest the age-related progressive neurodegeneration seen in spinocerebellar ataxia type 1. The shared features include morphological alterations in climbing fiber (CF) innervation of Purkinje cells (PCs). To further investigate the ability of ataxin-1 (ATXN1) to impact CF/PC innervation, this study used morphological and functional approaches to examine CF/PC innervation during postnatal development in ATXN1[30Q]-D776 and ATXN1[82Q] cerebella. Notably, ATXN1[30Q]-D776 induced morphological alterations consistent with the development of the innervation of PCs by CFs being compromised, including a reduction of CF translocation along the PC dendritic tree, and decreased pruning of CF terminals from the PC soma. As previously shown for ATXN1[82Q], ATXN1[30Q]-D776 must enter the nucleus of PCs to induce these alterations. Experiments using conditional ATXN1[30Q]-D776 mice demonstrate that both the levels and specific timing of mutant ATXN1 expression are critical for alteration of the CF-PC synapse. Together these observations suggest that ATXN1, expressed exclusively in PCs, alters expression of a gene(s) in the postsynaptic PC that are critical for its innervation by CFs. To investigate whether ATXN1[30Q]-D776 curbs the progressive disease in ATXN1[82Q]-S776 mice, we crossed ATXN1[30Q]-D776 and ATXN1[82Q]-S776 mice and found that double transgenic mice developed progressive PC atrophy. Thus, the results also show that to develop progressive cerebellar degeneration requires expressing ATXN1 with an expanded polyglutamine tract.

A conserved eEF2 coding variant in SCA26 leads to loss of translational fidelity and increased susceptibility to proteostatic insult.

The autosomal dominant spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of disorders exhibiting cerebellar atrophy and Purkinje cell degeneration whose subtypes arise from 31 distinct genetic loci. Our group previously published the locus for SCA26 on chromosome 19p13.3. In this study, we performed targeted deep sequencing of the critical interval in order to identify candidate causative variants in individuals from the SCA26 family. We identified a single variant that co-segregates with the disease phenotype that produces a single amino acid substitution in eukaryotic elongation factor 2. This substitution, P596H, sits in a domain critical for maintaining reading frame during translation. The yeast equivalent, P580H EF2, demonstrated impaired translocation, detected as an increased rate of -1 programmed ribosomal frameshift read-through in a dual-luciferase assay for observing translational recoding. This substitution also results in a greater susceptibility to proteostatic disruption, as evidenced by a more robust activation of a reporter gene driven by unfolded protein response activation upon challenge with dithiothreitol or heat shock in our yeast model system. Our results present a compelling candidate mutation and mechanism for the pathogenesis of SCA26 and further support the role of proteostatic disruption in neurodegenerative diseases.

Non-ATG-initiated translation directed by microsatellite expansions.

Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.

Occult Amyloid-ß-Related Angiitis: Neuroimaging Findings at 1.5T, 3T, and 7T MRI.

Cerebral amyloid angiopathy (CAA) is a progressive neurodegenerative small vessel disease that is associated with intracranial hemorrhage and cognitive impairment in the elderly. The clinical and radiographic presentations have many overlapping features with vascular cognitive impairment, hemorrhagic stroke, and Alzheimer disease (AD). Amyloid-ß-related angiitis (ABRA) is a form of primary CNS vasculitis linked to CAA, with the development of spontaneous autoimmune inflammation against amyloid in the vessel wall with resultant vasculitis. The diagnosis of ABRA and CAA is important. ABRA is often fatal if untreated and requires prompt immunosuppression. Important medical therapies such as anticoagulation and antiamyloid agents for AD are contraindicated in CAA. Here, we present a biopsy-proved case of ABRA with underlying occult CAA. Initial 1.5T and 3T MR imaging did not suggest CAA per the Boston Criteria 2.0. ABRA was not included in the differential diagnosis due to the lack of any CAA-related findings on conventional MR imaging. However, a follow-up 7T MR imaging revealed extensive cortical/subcortical cerebral microbleeds, cortical superficial siderosis, and intragyral hemorrhage in extensive detail throughout the supratentorial brain regions, which radiologically supported the diagnosis of ABRA in the setting of CAA. This case suggests an increased utility of high-field MR imaging to detect occult hemorrhagic neuroimaging findings with the potential to both diagnose more patients with CAA and diagnose them earlier.

A rare case of isolated IgG4-related hypophysitis with Rathke's cleft cyst presenting as panhypopituitarism.

Summary: We report a rare case of biopsy-proven isolated immunoglobulin G4 (IgG4)-related hypophysitis and Rathke's cleft cyst (RCC) presenting as panhypopituitarism. A 54-year-old Caucasian female presented with symptoms of slurred speech, altered mental status, polyuria and polydipsia and was found to have panhypopituitarism. Brain MRI showed a suprasellar mass with suspected intralesional hemorrhage. She underwent trans-sphenoidal resection due to MRI evidence of compression of the optic chiasm and left optic nerve. Preoperatively, she was started on hydrocortisone, levothyroxine and desmopressin. Histopathology demonstrated a RCC with adjacent lymphoplasmacytic hypophysitis with numerous IgG4-immunoreactive plasma cells. Hydrocortisone was stopped at 10 months after confirming hypothalamic-pituitary-adrenal (HPA)-axis recovery and desmopressin was stopped at 1 year. There was recurrence of a cystic mass at 1 year follow-up. Over 4 years of follow-up, she continued to require levothyroxine, and the mass remained stable in size. In order to begin to understand how this case's unique histopathological presentation influences clinical presentation, pituitary imaging and prognosis, we present an accompanying literature review. Learning points: Isolated IgG4 hypophysitis and coexisting Rathke's cleft cyst is a rare condition, which presents a diagnostic challenge. Recognizing its characteristic features can assist with early recognition and initiation of therapy to promote optimal outcomes. Further studies investigating the mechanisms promoting co-occurrence of these entities and their effect on prognosis are needed.

The Neuropathology of Autoimmune Ataxias.

Autoimmune-mediated ataxia has been associated with paraneoplastic disease, gluten enteropathy, Hashimoto thyroiditis as well as autoimmune disorders without a known associated disease. There have been relatively few reports describing the neuropathology of these conditions. This review is an attempt to consolidate those reports and determine the ways in which autoimmune ataxias can be neuropathologically differentiated from hereditary or other sporadic ataxias. In most instances, particularly in paraneoplastic forms, the presence of inflammatory infiltrates is a strong indicator of autoimmune disease, but it was not a consistent finding in all reported cases. Therefore, clinical and laboratory findings are important for assessing an autoimmune mechanism. Such factors as rapid rate of clinical progression, presence of known autoantibodies or the presence of a malignant neoplasm or other autoimmune disease processes need to be considered, particularly in cases where inflammatory changes are minimal or absent and the pathology is largely confined to the cerebellum and its connections, where the disease can mimic hereditary or other sporadic ataxias.

Comprehensive BCMA Expression Profiling in Adult Normal Human Brain Suggests a Low Risk of On-target Neurotoxicity in BCMA-targeting Multiple Myeloma Therapy.

B-cell maturation antigen (BCMA) is a target for the treatment of multiple myeloma with cytolytic therapies, such as chimeric antigen receptor T-cells or T-cell redirecting antibodies. To better understand the potential for "on-target/off-tumor" toxicity caused by BCMA-targeting cytolytic therapies in the brain, we investigated normal brain BCMA expression. An immunohistochemistry (IHC) assay using the E6D7B commercial monoclonal antibody was applied to 107 formalin-fixed, paraffin-embedded brain samples (cerebrum, basal ganglia, cerebellum, brainstem; 63 unique donors). Although immunoreactivity was observed in a small number of neurons in brain regions including the striatum, thalamus, midbrain, and medulla, this immunoreactivity was considered nonspecific and not reflective of BCMA expression because it was distinct from the membranous and Golgi-like pattern seen in positive control samples, was not replicated when a different IHC antibody (D6 clone) was used, and was not corroborated by in situ hybridization data. Analysis of RNA-sequencing data from 478 donors in the GTEx and Allen BrainSpan databases demonstrated low levels of BCMA RNA expression in the striatum of young donors with levels becoming negligible beyond 30 years of age. We concluded that BCMA protein is not present in normal adult human brain, and therefore on-target toxicity in the brain is unlikely.

Noninvasive detection of presymptomatic and progressive neurodegeneration in a mouse model of spinocerebellar ataxia type 1.

Recent studies with a conditional mouse model of spinocerebellar ataxia type 1 (SCA1) suggest that neuronal dysfunction is reversible and neurodegeneration preventable with early interventions. Success of such interventions will depend on early detection of neuronal and glial abnormalities before cell loss and availability of objective methods to monitor progressive neurodegeneration. Cerebellar concentrations of N-acetylaspartate (NAA), myo-inositol, and glutamate as measured by magnetic resonance spectroscopy (MRS) correlate with ataxia scores of patients with SCA1, indicating their potential as reliable biomarkers of neurodegeneration. Here we investigated whether neurochemical levels are altered by early, presymptomatic disease and whether they gauge disease progression in a mouse model of SCA1. Cerebellar neurochemical profiles of transgenic mice that overexpress the mutant human ataxin-1 (the SCA1[82Q] line) were measured longitudinally up to 1 year by MRS at 9.4 T and compared to those of transgenic mice that overexpress the normal human ataxin-1 (the SCA1[30Q] line) and wild-type controls. Multiple neurochemicals distinguished the SCA1[82Q] mice from controls with no overlap at all ages. Six neurochemicals were significantly different in SCA1[82Q] mice at 6 weeks, before major pathological and neurological changes. Alterations in NAA, myo-inositol, and glutamate progressively worsened and were significantly correlated (p < 0.0001) with disease progression as assessed by histology (molecular layer thickness and an overall severity score). Therefore, the neurochemicals that correlate with clinical status in patients reflected progressive pathology in the mouse model. These data demonstrate that presymptomatic and progressive neurodegeneration in SCA1 can be noninvasively monitored using MRS.

Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids May Extend above and below Pons and Is Associated with Other Autoimmune Diseases.

Many autoimmune diseases can affect the central nervous system, and their varying clinical presentations often confound a straightforward diagnosis. In this report, we describe a unique presentation of CLIPPERS syndrome. To our knowledge, this is the first case to demonstrate significant supratentorial involvement with symmetric and non-confluent lesions in the medial orbitofrontal cortex; additionally, this is the second case to describe an association between diagnoses of hypothyroidism and CLIPPERS.

Neurochemical alterations in spinocerebellar ataxia type 1 and their correlations with clinical status.

Robust biomarkers of neurodegeneration are critical for testing of neuroprotective therapies. The clinical applicability of such biomarkers requires sufficient sensitivity to detect disease in individuals. Here we tested the sensitivity of high field (4 tesla) proton magnetic resonance spectroscopy ((1)H MRS) to neurochemical alterations in the cerebellum and brainstem in spinocerebellar ataxia type 1 (SCA1). We measured neurochemical profiles that consisted of 10 to 15 metabolite concentrations in the vermis, cerebellar hemispheres and pons of patients with SCA1 (N = 9) and healthy controls (N = 15). Total NAA (N-acetylaspartate + N-acetylaspartylglutamate, tNAA) and glutamate were lower and glutamine, myo-inositol and total creatine (creatine + phosphocreatine, tCr) were higher in patients relative to controls, consistent with neuronal dysfunction/loss, gliotic activity, and alterations in glutamate-glutamine cycling and energy metabolism. Changes in tNAA, tCr, myo-inositol, and glutamate levels were discernible in individual spectra and the tNAA/myo-inositol ratio in the cerebellar hemispheres and pons differentiated the patients from controls with 100% specificity and sensitivity. In addition, tNAA, myo-inositol, and glutamate levels in the cerebellar hemispheres and the tNAA and myo-inositol levels in the pons correlated with ataxia scores (Scale for the Assessment and Rating of Ataxia, SARA). Two other biomarkers measured in the cerebrospinal fluid (CSF) of a subset of the volunteers (F(2)-isoprostanes asa marker of oxidative stress and glial fibrillary acidic protein (GFAP) as a marker of gliosis) were not different between patients and controls. These data demonstrate that (1)H MRS biomarkers can be utilized to noninvasively assess neuronal and glial status in individual ataxia patients.

Tract-specific analysis improves sensitivity of spinal cord diffusion MRI to cross-sectional and longitudinal changes in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease that causes progressive degeneration of motor neurons in the brain and the spinal cord. Corticospinal tract degeneration is a defining feature of ALS. However, there have been very few longitudinal, controlled studies assessing diffusion MRI (dMRI) metrics in different fiber tracts along the spinal cord in general or the corticospinal tract in particular. Here we demonstrate that a tract-specific analysis, with segmentation of ascending and descending tracts in the spinal cord white matter, substantially increases the sensitivity of dMRI to disease-related changes in ALS. Our work also identifies the tracts and spinal levels affected in ALS, supporting electrophysiologic and pathologic evidence of involvement of sensory pathways in ALS. We note changes in diffusion metrics and cord cross-sectional area, with enhanced sensitivity to disease effects through a multimodal analysis, and with strong correlations between these metrics and spinal components of ALSFRS-R.

Precision Targeted Ablation of Fine Neurovascular Structures In Vivo Using Dual-mode Ultrasound Arrays.

Carotid bodies (CBs) are chemoreceptors that monitor and register changes in the blood, including the levels of oxygen, carbon dioxide, and pH, and regulate breathing. Enhanced activity of CBs was shown to correlate with a significant elevation in the blood pressure of patients with hypertension. CB removal or denervation were previously shown to reduce hypertension. Here we demonstrate the feasibility of a dual-mode ultrasound array (DMUA) system to safely ablate the CB in vivo in a spontaneously hypertensive rat (SHR) model of hypertension. DMUA imaging was used for guiding and monitoring focused ultrasound (FUS) energy delivered to the target region. In particular, 3D imaging was used to identify the carotid bifurcation for targeting the CBs. Intermittent, high frame rate imaging during image-guided FUS (IgFUS) delivery was used for monitoring the lesion formation. DMUA imaging provided feedback for closed-loop control (CLC) of the lesion formation process to avoid overexposure. The procedure was tolerated well in over 100 SHR and normotensive rats that received unilateral and bilateral treatments. The measured mean arterial pressure (MAP) exhibited measurable deviation from baseline 2-4 weeks post IgFUS treatment. The results suggest that the direct unilateral FUS treatment of the CB might be sufficient to reduce the blood pressure in hypertensive rats and justify further investigation in large animals and eventually in human patients.