Ruben Smith

Synaptic dysfunction in Huntington's disease: a new perspective.

Abstract.Huntington’s disease (HD) is caused by a polyglutamine expansion in the protein huntingtin and is characterized by intraneuronal inclusions and widespread neuronal death at the late stage of the disease. In research, most of the emphasis has been on understanding the cell death and its mechanisms. Until recently, it was believed that the vast majority, if not all, of the symptoms in HD are a direct consequence of neurodegeneration. However, increasing evidence shows that subtle alterations in synaptic function could underlie the early symptoms. It is of particular interest to understand the nature of this neuronal dysfunction. Normal huntingtin interacts with various cytoskeletal and synaptic vesicle proteins that are essential for exocytosis and endocytosis. Altered interactions of mutant huntingtin with its associated partners could contribute to abnormal synaptic transmission in HD. This review describes recent advances in understanding synaptic dysfunction in HD.

Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment

Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntingtons disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt.

Loss of SNAP-25 and rabphilin 3a in sensory-motor cortex in Huntington’s disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG‐expansion in the gene encoding the protein huntingtin. The disease is characterized by progressive motor disturbances, cognitive defects, dementia, and weight loss. Using western blotting and immunohistochemistry we have assessed the expression levels and patterns of a number of proteins involved in neurotransmitter release in post‐mortem frontal cortex samples from 10 HD cases with different disease grades. We report a loss of the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) protein, synaptosome‐associated protein 25 (SNAP 25) in HD brains of grades I–IV. Moreover, in brains of grade III and IV we found a reduction in rabphilin 3a, a protein involved in vesicle docking and recycling. These losses appear to be specific and not due to a general loss of synapses in the HD cortex. Thus, levels of synaptobrevin II, syntaxin 1, rab3a or synaptophysin are unaltered in the same patient samples. SNAP 25 and rabphilin 3a are crucial for neurotransmitter release. Therefore, we suggest that a deficient pre‐synaptic transmitter release may underlie some of the symptoms of HD.

18F-AV-1451 tau PET imaging correlates strongly with tau neuropathology in MAPT mutation carriers

Little is known about how the in vivo tau PET signal relates to post-mortem tau neuropathology. Smith et al. provide the first evidence that the two are highly correlated by showing that the tau PET tracer 18F-AV-1451 accurately detects tau pathology in subjects with mutations in the tau (MAPT) gene.

Increased basal ganglia binding of 18F‐AV‐1451 in patients with progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is difficult to diagnose accurately. The recently developed tau PET tracers may improve the diagnostic work‐up of PSP.

Mutant huntingtin interacts with β-tubulin and disrupts vesicular transport and insulin secretion

Huntingtons disease is a severe progressive neurodegenerative disorder caused by a CAG expansion in the IT15 gene, which encodes huntingtin. The disease primarily affects the neostriatum and cerebral cortex and also associates with increased incidence of diabetes. Here, we show that mutant huntingtin disrupts intracellular transport and insulin secretion by direct interference with microtubular beta-tubulin. We demonstrate that mutant huntingtin impairs glucose-stimulated insulin secretion in insulin-producing beta-cells, without altering stored levels of insulin. Using VSVG-YFP, we show that mutant huntingtin retards post-Golgi transport. Moreover, we demonstrate that the speed of insulin vesicle trafficking is reduced. Using immunoprecipitation of mutant and wild-type huntingtin in combination with mass spectrometry, we reveal an enhanced and aberrant interaction between mutant huntingtin and beta-tubulin, implying the underlying mechanism of impaired intracellular transport. Thus, our findings have revealed a novel pathogenetic process by which mutant huntingtin may disrupt hormone exocytosis from beta-cells and possibly impair vesicular transport in any cell that expresses the pathogenic protein.

The role of pallidal serotonergic function in Parkinson's disease dyskinesias: a positron emission tomography study.

We have investigated the role of globus pallidus (GP) serotonergic terminals in the development of levodopa-induced dyskinesias (LIDs) in Parkinsons disease (PD). We studied 12 PD patients without LIDs, 12 PD patients with LIDs, and 12 healthy control subjects. We used (11)C-DASB positron emission tomography (PET), a marker of serotonin transporter availability, and (11)C-raclopride PET to measure changes in synaptic dopamine levels following levodopa administration. PD patients without LIDs showed a significant reduction of GP serotonin transporter binding compared with healthy controls although this was within the normal range in PD patients with LIDs. Levels of GP serotonin transporter binding correlated positively with severity of dyskinesias. (11)C-raclopride PET detected a significant rise in GP synaptic dopamine levels of patients with LIDs after a levodopa challenge but not in patients with a stable response. Our findings indicate that LIDs in PD are associated with higher GP serotonergic function. This increased serotonin function may result in further dysregulation of thalamocortical signals and so promote the expression of dyskinesias.

Tyrosine hydroxylase expression is unstable in a human immortalized mesencephalic cell line--studies in vitro and after intracerebral grafting in vivo.

We have studied the stability of the dopaminergic phenotype in a conditionally immortalized human mesencephalic cell line, MESC2.10. Even though MESC2.10 cells exhibit features of dopaminergic neurons in vitro, none of the cells expressed tyrosine hydroxylase (TH) after transplantation into a rat model of Parkinsons disease. We examined whether this is caused by cell death or loss of transmitter phenotype. Cells were cultured in differentiation medium, then harvested and replated into the same medium where they continued to express TH, whereas replated cells fed medium lacking differentiation factors (dibutyryl cAMP and glial cell line-derived neurotrophic factor) did not. Interestingly, cultures grown in the absence of differentiation factors could regain TH expression once exposed to differentiation medium. Our data suggest that TH expression in vitro is inducible in neurons derived from the MESC2.10 cell line and that the dopaminergic phenotype of these cells in vivo might be unstable.

Depletion of rabphilin 3A in a transgenic mouse model (R6/1) of Huntington's disease, a possible culprit in synaptic dysfunction.

Huntingtons disease (HD) is a hereditary neurodegenerative disorder characterized by progressive psychiatric, cognitive, and motor disturbances. We studied the expression of synaptic vesicle proteins in the R6/1 transgenic mouse model of HD. We observed that the levels of rabphilin 3A, a protein involved in exocytosis, is substantially decreased in synapses of most brain regions in R6/1 mice. The appearance of the reduction coincides with the onset of motor deficits and behavioral disturbances. Double immunohistochemistry did not show colocalization between rabphilin 3A and huntingtin aggregates in the HD mice. Using in situ hybridization, we demonstrated that rabphilin 3A mRNA expression was substantially reduced in the R6/1 mouse cortex compared to wild-type mice. Our results indicate that a decrease in mRNA levels underlie the depletion of protein levels of rabphilin 3A, and we suggest that this reduction may be involved in causing impaired synaptic transmission in R6/1 mice.

Tau neuropathology correlates with FDG-PET, but not AV-1451-PET, in progressive supranuclear palsy

not shown convincing binding of AV-1451 to tau pathology in PSP. We have recently shown increased retention in vivo of F-AV-1451 in the basal ganglia, but not in the cortex, of patients with PSP [8]. Here, we present data from a 71-year-old male subject, who was diagnosed with PSP in 2011 after a 2-year history of increasing bradykinesia, rigidity, and falls (see Supplementary material for detail). Seven months prior to death the subject was scanned with F-AV-1451 PET, F-AV-FDG-PET, and MRI (Fig. 1a–d). F-AV-1451 PET showed retention in the basal ganglia, but not cortex (Fig. 1a, c). F-AV-FDG-PET revealed decreased metabolism in the frontal cortex and basal ganglia (Fig. 1b). Voxel-based morphometry showed cortical atrophy, predominantly in the frontal cortex (Fig. 1d). For methodological data, see the supplement. Post mortem neuropathological assessment confirmed the diagnosis of PSP, with a marked atrophy of the midbrain and a frontal cortical atrophy. Gallyas silver stain showed dense neurites and stained cell bodies in the basal ganglia, midbrain, and cerebral cortex, as well as cortical tufted astrocytes. Immunohistochemistry showed positivity for 4R tau inclusions, but no 3R tau staining (see supplement for method). Amyloid stains as well as α-synuclein immunohistochemistry were negative (data not shown). Tau-positive (AT8 immunohistochemistry) neurites and cell bodies were quantified using unbiased stereology (see supplement and [7] for methods), and results were correlated with F-AV-1451 and F-FDG retention in the same regions (Fig. 1 ). In the frontal, temporal, and parietal cortex and subcortical white matter, there was widespread tau pathology, whereas the occipital lobe and cerebellar grey matter were relatively spared (Fig. 1e–p). The basal ganglia and mesencephalon showed a widespread and intense tau pathology using immunohistochemistry. Comparing the F-AV-1451 retention to neurite density, there was no correlation between PET signal Radiotracers for tau have recently become available for positron emission tomography (PET) studies in neurodegenerative disorders. The currently most utilized tracer FAV-1451 binds to paired helical filaments containing both 3R and 4R tau in Alzheimer’s disease (AD) in vitro [3, 4, 6]. Furthermore, F-AV-1451 can quantify AD-like tau pathology in MAPT R406W mutation carriers in vivo [7]. In corticobasal degeneration, there is a clear binding to 4R tau pathology, but SUVRs are generally lower than in AD [2, 5]. Whether the tracer binds to the straight filament 4R pathology in progressive supranuclear palsy (PSP) is not fully established. Autoradiographic studies [3, 4, 6, 8] have