Correction: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3'-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.1004749.].

Tissue Iron in Friedreich Ataxia.

Heart, dentate nucleus, and dorsal root ganglia (DRG) are targets of tissue damage in Friedreich ataxia (FA). This report summarizes the histology and histopathology of iron in the main tissues affected by FA. None of the affected anatomical sites reveals an elevation of total iron levels. In the myocardium, a small percentage of fibers shows iron-reactive granular inclusions. The accumulation of larger iron aggregates and fiber invasion cause necrosis and damage to the contractile apparatus. In the dentate nucleus, the principal FA-caused tissue injury is neuronal atrophy and grumose reaction. X-ray fluorescence mapping of iron in the dentate nucleus in FA shows retention of the metal in the center of the collapsed structure. Immunohistochemistry of ferritin, a surrogate marker of tissue iron, confirms strong expression in oligodendrocytes of the efferent white matter of the dentate nucleus and abundance of ferritin-positive microglia in the atrophic gray matter. Iron dysmetabolism in DRG is complex and consists of prominent expression of ferritin in hyperplastic satellite cells and residual nodules, also a loss of the iron export protein ferroportin from the cytoplasm of the remaining DRG nerve cells.

Contextualizing the pathology in the essential tremor cerebellar cortex: a patholog-omics approach.

Several morphological changes, centered in/around Purkinje cells (PCs), have been identified in the cerebellum of essential tremor (ET) patients. These changes have not been contextualized within a broader degenerative disease spectrum, limiting their interpretability. To address this, we compared the severity and patterning of degenerative changes within the cerebellar cortex in patients with ET, other neurodegenerative disorders of the cerebellum (spinocerebellar ataxias (SCAs), multiple system atrophy (MSA)], and other disorders that may involve the cerebellum [Parkinson's disease (PD), dystonia]. Using a postmortem series of 156 brains [50 ET, 23 SCA (6 SCA3; 17 SCA 1, 2 or 6), 15 MSA, 29 PD, 14 dystonia, 25 controls], we generated data on 37 quantitative morphologic metrics, which were grouped into 8 broad categories: (1) PC loss, (2) heterotopic PCs, (3) PC dendritic changes, (4) PC axonal changes (torpedoes), (5) PC axonal changes (other than torpedoes), (6) PC axonal changes (torpedo-associated), (7) basket cell axonal hypertrophy, (8) climbing fiber-PC synaptic changes. Our analyses used z scored raw data for each metric across all diagnoses (5772 total data items). Principal component analysis revealed that diagnostic groups were not uniform with respect to cerebellar pathology. Dystonia and PD each differed from controls in only 2/37 metrics, whereas ET differed in 21, SCA3 in 8, MSA in 19, and SCA1/2/6 in 26 metrics. Comparing ET with primary disorders of cerebellar degeneration (i.e., SCAs), we observed a spectrum of changes reflecting differences of degree, being generally mild in ET and SCA3 and more severe in SCA1/2/6. Comparative analyses across morphologic categories demonstrated differences in relative expression, defining distinctive patterns of changes in these groups. Thus, the degree of cerebellar degeneration in ET aligns it with a milder end in the spectrum of cerebellar degenerative disorders, and a somewhat distinctive signature of degenerative changes marks each of these disorders.

The Neuropathology of Spinocerebellar Ataxia Type 3/Machado-Joseph Disease.

Spinocerebellar ataxia type 3 (SCA-3)/Machado-Joseph disease (MJD), the most common autosomal dominant ataxia, affects many regions of the brain and spinal cord. Similar to SCA-1, SCA-2, SCA-6, SCA-7, and SCA-17, the mutation consists of a pathogenic translated cytosine-adenine-guanine (CAG) trinucleotide repeat expansion. Almost invariably, the substantia nigra and the dentate nucleus of the cerebellum bear the brunt of the disease, and these lesions account for the Parkinsonian and ataxic phenotypes. Lesions of motor nuclei in the brain stem cause the complex disturbance of ocular motility and weakness of the tongue. Atrophy of the basis pontis is common, and polyglutamine-positive neuronal intranuclear inclusion bodies are most readily found in the pontine gray. Abnormalities of basal ganglia, thalamus, spinal cord, dorsal root ganglia, and sensory peripheral nerves are more variable. This report of the main neuropathological lesions is based on the study of 12 genetically confirmed autopsy cases of SCA-3/MJD. In the cerebellum, all layers of the cortex remain normal, but the dentate nucleus exhibits neuronal loss and a peculiar proliferation of synaptic terminals termed grumose regeneration. The clusters surrounding residual neuronal cell bodies and dendrites are interpreted as a response to loss of γ-aminobutyric acid (GABA)-A-receptors and lack of gephyrin, a protein that accomplishes the proper positioning of GABA-A- and glycine receptors. At the spinal level, dorsal root ganglia reveal proliferation of satellite cells, active neuronal destruction, and residual nodules. The spinal cord shows total or subtotal loss of neurons in the dorsal nuclei, anterior horn cell atrophy, and variable long tract degeneration. While misfolding of ataxin-3 due to overly long polyglutamine stretches is a critical contributor to the pathogenesis of SCA-3/MJD, the great neuropathological complexity of the disorder remains largely unexplained.

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia type 3 pathogenesis.

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

ATXN2-AS, a gene antisense to ATXN2, is associated with spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis.

OBJECTIVE: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by a CAG repeat expansion in the gene ataxin-2 (ATXN2). ATXN2 intermediate-length CAG expansions were identified as a risk factor for amyotrophic lateral sclerosis (ALS). The ATXN2 CAG repeat is translated into polyglutamine, and SCA2 pathogenesis has been thought to derive from ATXN2 protein containing an expanded polyglutamine tract. However, recent evidence of bidirectional transcription at multiple CAG/CTG disease loci has led us to test whether additional mechanisms of pathogenesis may contribute to SCA2. METHODS: In this work, using human postmortem tissue, various cell models, and animal models, we provide the first evidence that an antisense transcript at the SCA2 locus contributes to SCA2 pathogenesis. RESULTS: We demonstrate the expression of a transcript, containing the repeat as a CUG tract, derived from a gene (ATXN2-AS) directly antisense to ATXN2. ATXN2-AS transcripts with normal and expanded CUG repeats are expressed in human postmortem SCA2 brains, human SCA2 fibroblasts, induced SCA2 pluripotent stem cells, SCA2 neural stem cells, and lymphoblastoid lines containing an expanded ATXN2 allele associated with ALS. ATXN2-AS transcripts with a CUG repeat expansion are toxic in an SCA2 cell model and form RNA foci in SCA2 cerebellar Purkinje cells. Finally, we detected missplicing of amyloid beta precursor protein and N-methyl-D-aspartate receptor 1 in SCA2 brains, consistent with findings in other diseases characterized by RNA-mediated pathogenesis. INTERPRETATION: These results suggest that ATXN2-AS has a role in SCA2 and possibly ALS pathogenesis, and may therefore provide a novel therapeutic target for these diseases. Ann Neurol 2016;80:600-615.

Abnormal climbing fibre-Purkinje cell synaptic connections in the essential tremor cerebellum.

Structural changes in Purkinje cells have been identified in the essential tremor cerebellum, although the mechanisms that underlie these changes remain poorly understood. Climbing fibres provide one of the major excitatory inputs to Purkinje cells, and climbing fibre-Purkinje cell connections are essential for normal cerebellar-mediated motor control. The distribution of climbing fibre-Purkinje cell synapses on Purkinje cell dendrites is dynamically regulated and may be altered in disease states. The aim of the present study was to examine the density and distribution of climbing fibre-Purkinje cell synapses using post-mortem cerebellar tissue of essential tremor cases and controls. Using vesicular glutamate transporter type 2 immunohistochemistry, we labelled climbing fibre-Purkinje cell synapses of 12 essential tremor cases and 13 age-matched controls from the New York Brain Bank. Normally, climbing fibres form synapses mainly on the thick, proximal Purkinje cell dendrites in the inner portion of the molecular layer, whereas parallel fibres form synapses on the thin, distal Purkinje cell spiny branchlets. We observed that, compared with controls, essential tremor cases had decreased climbing fibre-Purkinje cell synaptic density, more climbing fibres extending to the outer portion of the molecular layer, and more climbing fibre-Purkinje cell synapses on the thin Purkinje cell spiny branchlets. Interestingly, in essential tremor, the increased distribution of climbing fibre-Purkinje cell synapses on the thin Purkinje cell branchlets was inversely associated with clinical tremor severity, indicating a close relationship between the altered distribution of climbing fibre-Purkinje cell connections and tremor. These findings suggest that abnormal climbing fibre-Purkinje cell connections could be of importance in the pathogenesis of essential tremor.

A novel transgenic rat model for spinocerebellar ataxia type 17 recapitulates neuropathological changes and supplies in vivo imaging biomarkers.

Spinocerebellar ataxia 17 (SCA17) is an autosomal-dominant, late-onset neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat in the TATA-box-binding protein (TBP). To further investigate this devastating disease, we sought to create a first transgenic rat model for SCA17 that carries a full human cDNA fragment of the TBP gene with 64 CAA/CAG repeats (TBPQ64). In line with previous observations in mouse models for SCA17, TBPQ64 rats show a severe neurological phenotype including ataxia, impairment of postural reflexes, and hyperactivity in early stages followed by reduced activity, loss of body weight, and early death. Neuropathologically, the severe phenotype of SCA17 rats was associated with neuronal loss, particularly in the cerebellum. Degeneration of Purkinje, basket, and stellate cells, changes in the morphology of the dendrites, nuclear TBP-positive immunoreactivity, and axonal torpedos were readily found by light and electron microscopy. While some of these changes are well recapitulated in existing mouse models for SCA17, we provide evidence that some crucial characteristics of SCA17 are better mirrored in TBPQ64 rats. Thus, this SCA17 model represents a valuable tool to pursue experimentation and therapeutic approaches that may be difficult or impossible to perform with SCA17 transgenic mice. We show for the first time positron emission tomography (PET) and diffusion tensor imaging (DTI) data of a SCA animal model that replicate recent PET studies in human SCA17 patients. Our results also confirm that DTI are potentially useful correlates of neuropathological changes in TBPQ64 rats and raise hope that DTI imaging could provide a biomarker for SCA17 patients.

A GGC-repeat expansion in ZFHX3 encoding polyglycine causes spinocerebellar ataxia type 4 and impairs autophagy.

Despite linkage to chromosome 16q in 1996, the mutation causing spinocerebellar ataxia type 4 (SCA4), a late-onset sensory and cerebellar ataxia, remained unknown. Here, using long-read single-strand whole-genome sequencing (LR-GS), we identified a heterozygous GGC-repeat expansion in a large Utah pedigree encoding polyglycine (polyG) in zinc finger homeobox protein 3 (ZFHX3), also known as AT-binding transcription factor 1 (ATBF1). We queried 6,495 genome sequencing datasets and identified the repeat expansion in seven additional pedigrees. Ultrarare DNA variants near the repeat expansion indicate a common distant founder event in Sweden. Intranuclear ZFHX3-p62-ubiquitin aggregates were abundant in SCA4 basis pontis neurons. In fibroblasts and induced pluripotent stem cells, the GGC expansion led to increased ZFHX3 protein levels and abnormal autophagy, which were normalized with small interfering RNA-mediated ZFHX3 knockdown in both cell types. Improving autophagy points to a therapeutic avenue for this novel polyG disease. The coding GGC-repeat expansion in an extremely G+C-rich region was not detectable by short-read whole-exome sequencing, which demonstrates the power of LR-GS for variant discovery.

Nikolaus Friedreich and degenerative atrophy of the dorsal columns of the spinal cord.

Nikolaus Friedreich (1825-1882) presented clinical findings in six patients with a severe hereditary disorder of the nervous system and secured full autopsies in four of them. He was fascinated by the spinal cord lesions in the siblings of two unrelated families, and in the first three of his five long articles stressed the destruction of the dorsal columns. He recognized the relatively minor symmetrical lesions of the anterolateral fasciculi but did not separate dorsal spinocerebellar tracts (Flechsig's bundles) and corticospinal tracts. Although he studied the dorsal spinal roots in great detail and established their principal abnormality, namely, axonal thinning without axonal loss, he reported dorsal root ganglia as entirely normal. He made an insightful description of atrophic neurons in the gracile nuclei (clavae) but overlooked the invariable atrophy of the dentate nuclei. He followed the families over a period of 14 years, but acknowledged the hereditary nature of the disease only very late. He proposed a developmental defect for the medulla oblongata, retaining his interpretation that the spinal lesion was inflammatory. This review honors Friedreich for his insight into a 'new' disease in the late 19th century and updates his neuropathological findings. It is remarkable that Friedreich also described the abnormal hearts in the disease that now bears his name since hypertrophic cardiomyopathy is now recognized as the main cause of death in Friedreich's ataxia.

Central role and mechanisms of ß-cell dysfunction and death in friedreich ataxia-associated diabetes.

OBJECTIVE: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused in almost all cases by homozygosity for a GAA trinucleotide repeat expansion in the frataxin gene. Frataxin is a mitochondrial protein involved in iron homeostasis. FRDA patients have a high prevalence of diabetes, the pathogenesis of which is not known. We aimed to evaluate the relative contribution of insulin resistance and ß-cell failure and the pathogenic mechanisms involved in FRDA diabetes. METHODS: Forty-one FRDA patients, 26 heterozygous carriers of a GAA expansion, and 53 controls underwent oral and intravenous glucose tolerance tests. ß-Cell proportion was quantified in postmortem pancreas sections from 9 unrelated FRDA patients. Using an in vitro disease model, we studied how frataxin deficiency affects ß-cell function and survival. RESULTS: FRDA patients had increased abdominal fat and were insulin resistant. This was not compensated for by increased insulin secretion, resulting in a markedly reduced disposition index, indicative of pancreatic ß-cell failure. Loss of glucose tolerance was driven by ß-cell dysfunction, which correlated with abdominal fatness. In postmortem pancreas sections, pancreatic islets of FRDA patients had a lower ß-cell content. RNA interference-mediated frataxin knockdown impaired glucose-stimulated insulin secretion and induced apoptosis in rat ß cells and human islets. Frataxin deficiency sensitized ß cells to oleate-induced and endoplasmic reticulum stress-induced apoptosis, which could be prevented by the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. INTERPRETATION: Pancreatic ß-cell dysfunction is central to diabetes development in FRDA as a result of mitochondrial dysfunction and higher sensitivity to metabolic and endoplasmic reticulum stress-induced ß-cell death.

The reciprocal cerebellar circuitry in human hereditary ataxia.

Clinicoanatomic correlation in the spinocerebellar ataxias (SCA) and Friedreich's ataxia (FRDA) is difficult as these diseases differentially affect multiple sites in the central and peripheral nervous systems. A new way to study cerebellar ataxia is the systematic analysis of the "reciprocal cerebellar circuitry" that consists of tightly organized reciprocal connections between Purkinje cells, dentate nuclei (DN), and inferior olivary nuclei (ION). This circuitry is similar to but not identical with the "cerebellar module" in experimental animals. Neurohumoral transmitters operating in the circuitry are both inhibitory (γ-aminobutyric acid in corticonuclear and dentato-olivary fibers) and excitatory (glutamate in olivocerebellar or climbing fibers). Glutamatergic climbing fibers also issue collaterals to the DN. The present study applied five immunohistochemical markers in six types of SCA (1, 2, 3, 6, 7, 17), genetically undefined SCA, FRDA, and FRDA carriers to identify interruptions within the circuitry: calbindin-D28k, neuron-specific enolase, glutamic acid decarboxylase, and vesicular glutamate transporters 1 and 2. Lesions of the cerebellar cortex, DN, and ION were scored according to a guide as 0 (normal), 1 (mild), 2 (moderate), and 3 (severe). Results of each of the five immunohistochemical stains were examined separately for each of the three regions. Combining scores of each anatomical region and each stain yielded a total score as an indicator of pathological severity. Total scores ranged from 16 to 38 in SCA-1 (nine cases); 22 to 39 in SCA-2 (six cases); 9 to 15 in SCA-3 (four cases); and 13 and 25 in SCA-6 (two cases). In single cases of SCA-7 and SCA-17, scores were 16 and 31, respectively. In two genetically undefined SCA, scores were 36 and 37, respectively. In nine cases of FRDA, total scores ranged from 11 to 19. The low scores in SCA-3 and FRDA reflect selective atrophy of the DN. The FRDA carriers did not differ from normal controls. These observations offer a semiquantitative assessment of the critical role of the DN in the ataxic phenotype of SCA and FRDA while other parts of the circuitry appear less important.

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

Friedreich's ataxia causes redistribution of iron, copper, and zinc in the dentate nucleus.

Friedreich's ataxia (FRDA) causes selective atrophy of the large neurons of the dentate nucleus (DN). High iron (Fe) concentration and failure to clear the metal from the affected brain tissue are potential risk factors in the progression of the lesion. The DN also contains relatively high amounts of copper (Cu) and zinc (Zn), but the importance of these metals in FRDA has not been established. This report describes nondestructive quantitative X-ray fluorescence (XRF) and "mapping" of Fe, Cu, and Zn in polyethylene glycol-dimethylsulfoxide (PEG/DMSO)-embedded DN of 10 FRDA patients and 13 controls. Fe fluorescence arose predominantly from the hilar white matter, whereas Cu and Zn were present at peak levels in DN gray matter. Despite collapse of the DN in FRDA, the location of the peak Fe signal did not change. In contrast, the Cu and Zn regions broadened and overlapped extensively with the Fe-rich region. Maximal metal concentrations did not differ from normal (in micrograms per milliliter of solid PEG/DMSO as means ± S.D.): Fe normal, 364 ± 117, FRDA, 344 ± 159; Cu normal, 33 ± 13, FRDA, 33 ± 18; and Zn normal, 32 ± 16, FRDA, 33 ± 19. Tissues were recovered from PEG/DMSO and transferred into paraffin for matching with immunohistochemistry of neuron-specific enolase (NSE), glutamic acid decarboxylase (GAD), and ferritin. NSE and GAD reaction products confirmed neuronal atrophy and grumose degeneration that coincided with abnormally diffuse Cu and Zn zones. Ferritin immunohistochemistry matched Fe XRF maps, revealing the most abundant reaction product in oligodendroglia of the DN hilus. In FRDA, these cells were smaller and more numerous than normal. In the atrophic DN gray matter of FRDA, anti-ferritin labeled mostly hypertrophic microglia. Immunohistochemistry and immunofluorescence of the Cu-responsive proteins Cu,Zn-superoxide dismutase and Cu(++)-transporting ATPase α-peptide did not detect specific responses to Cu redistribution in FRDA. In contrast, metallothionein (MT)-positive processes were more abundant than normal and contributed to the gliosis of the DN. The isoforms of MT, MT-1/2, and brain-specific MT-3 displayed only limited co-localization with glial fibrillary acidic protein. The results suggest that MT can provide effective protection against endogenous Cu and Zn toxicity in FRDA, similar to the neuroprotective sequestration of Fe in holoferritin.

Overexpression of the autophagic beclin-1 protein clears mutant ataxin-3 and alleviates Machado-Joseph disease.

Machado-Joseph disease, also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 misfolding, intracellular accumulation of aggregates and neuronal degeneration. Here we investigated the implication of autophagy, the major pathway for organelle and protein turnover, in the accumulation of mutant ataxin-3 aggregates and neurodegeneration found in Machado-Joseph disease and we assessed whether specific stimulation of this pathway could mitigate the disease. Using tissue from patients with Machado-Joseph disease, transgenic mice and a lentiviral-based rat model, we found an abnormal expression of endogenous autophagic markers, accumulation of autophagosomes and decreased levels of beclin-1, a crucial protein in the early nucleation step of autophagy. Lentiviral vector-mediated overexpression of beclin-1 led to stimulation of autophagic flux, mutant ataxin-3 clearance and overall neuroprotective effects in neuronal cultures and in a lentiviral-based rat model of Machado-Joseph disease. These data demonstrate that autophagy is a key degradation pathway, with beclin-1 playing a significant role in alleviating Machado-Joseph disease pathogenesis.

The cerebellar component of Friedreich's ataxia.

Lack of frataxin in Friedreich's ataxia (FRDA) causes a complex neurological and pathological phenotype. Progressive atrophy of the dentate nucleus (DN) is a major intrinsic central nervous system lesion. Antibodies to neuron-specific enolase (NSE), calbindin, glutamic acid decarboxylase (GAD), and vesicular glutamate transporters 1 and 2 (VGluT1, VGluT2) allowed insight into the disturbed synaptic circuitry of the DN. The available case material included autopsy specimens of 24 patients with genetically defined FRDA and 14 normal controls. In FRDA, the cerebellar cortex revealed intact Purkinje cell somata and dendrites as assessed by calbindin immunoreactivity. The DN, however, displayed severe loss of large NSE-reactive neurons. Small neurons remained intact. Labeling of Purkinje cells, basket fibers, Golgi neurons, and Golgi axonal plexuses with antibodies to GAD indicated normal intrinsic circuitry of the cerebellar cortex involving γ-aminobutyric acid (GABA). In contrast, the DN displayed severe loss of GABA-ergic terminals and formation of GAD- and calbindin-reactive grumose degeneration. The surviving small GAD-positive DN neurons provided normal GABA-ergic terminals to intact inferior olivary nuclei. The olives also received normal glutamatergic terminals as shown by VGluT2-reactivity. VGluT1-immunocytochemistry of the cerebellar cortex confirmed normal glutamatergic input to the molecular layer by parallel fibers and the granular layer by mossy fibers. VGluT2-immunoreactivity visualized normal climbing fibers and mossy fiber terminals. The DN, however, showed depletion of VGluT1- and VGluT2-reactive terminals arising from climbing and mossy fiber collaterals. The main functional deficit underlying cerebellar ataxia in FRDA is defective processing of inhibitory and excitatory impulses that converge on the large neurons of the DN. The reason for the selective vulnerability of these nerve cells remains elusive.

The neuropathology of late-onset Friedreich's ataxia.

Friedreich's ataxia (FRDA) affects very young persons. In a large series, the mean ages of onset and death were 11 and 38 years, respectively. The clinical spectrum of FRDA has expanded after genetic confirmation of the mutation became a routine laboratory test. The main cause of death in juvenile-onset FRDA is cardiomyopathy whereas patients with late-onset are more likely to succumb to neurological disability or an intercurrent illness. Many patients with early onset now survive for 20 years or longer. This study made a systematic comparison of the neuropathology in 14 patients with juvenile onset and long survival, and five patients with late onset and long survival. Mean ages of onset (± standard deviation) were 10 ± 5 and 28 ± 13 years, respectively. Disease durations were 33 ± 11 and 47 ± 11 years, respectively. Cross-sectional areas of the thoracic spinal cord were greatly reduced from the normal state but did not differ between the two groups. Similarly, the neurons of dorsal root ganglia were significantly reduced in size in both juvenile- and late-onset cases of FRDA. The dentate nucleus showed severe loss of neurons as well as modification and destruction of corticonuclear terminals in all FRDA patients. Delayed atrophy of the dentate nucleus is the likely cause of the ataxic phenotype of FRDA in late-onset cases, but the reason for the delay is unknown. Frataxin levels in the dentate nucleus of two patients with late onset were similar to those of seven patients with juvenile onset.

Friedreich cardiomyopathy is a desminopathy.

Heart disease is an integral part of Friedreich ataxia (FA) and the most common cause of death in this autosomal recessive disease. The result of the mutation is lack of frataxin, a small mitochondrial protein. The clinical and pathological phenotypes of FA are complex, involving brain, spinal cord, dorsal root ganglia, sensory nerves, heart, and endocrine pancreas. The hypothesis is that frataxin deficiency causes downstream changes in the proteome of the affected tissues, including the heart. A proteomic analysis of heart proteins in FA cardiomyopathy by antibody microarray, Western blots, immunohistochemistry, and double-label laser scanning confocal immunofluorescence microscopy revealed upregulation of desmin and its chaperone protein, αB-crystallin. In normal hearts, these two proteins are co-localized at intercalated discs and Z discs. In FA, desmin and αB-crystallin aggregate, causing chaotic modification of intercalated discs, clustering of mitochondria, and destruction of the contractile apparatus of cardiomyocytes. Western blots of tissue lysates in FA cardiomyopathy reveal a truncated desmin isoprotein that migrates at a lower molecular weight range than wild type desmin. While desmin and αB-crystallin are not mutated in FA, the accumulation of these proteins in FA hearts allows the conclusion that FA cardiomyopathy is a desminopathy akin to desmin myopathy of skeletal muscle.