Karen Ma

Increased number of Purkinje cell dendritic swellings in essential tremor

Background and Purpose:  Essential Tremor (ET) is among the most prevalent neurologic disorders. Growing clinical and neuro‐imaging evidence implicates cerebellar dysfunction in the pathogenesis of ET and emerging postmortem studies have identified structural changes in the cerebellum, particularly in Purkinje cells. In this study we systematically quantified focal Purkinje cell dendritic swellings (DS) in 20 ET vs. 19 control brains.

Reduced Purkinje cell dendritic arborization and loss of dendritic spines in essential tremor

Based on accumulating post-mortem evidence of abnormalities in Purkinje cell biology in essential tremor, we hypothesized that regressive changes in dendritic morphology would be apparent in the Purkinje cell population in essential tremor cases versus age-matched controls. Cerebellar cortical tissue from 27 cases with essential tremor and 27 age-matched control subjects was processed by the Golgi-Kopsch method. Purkinje cell dendritic anatomy was quantified using a Neurolucida microscopic system interfaced with a motorized stage. In all measures, essential tremor cases demonstrated significant reductions in dendritic complexity compared with controls. Median values in essential tremor cases versus controls were: 5712.1 versus 10 403.2 µm (total dendrite length, P=0.01), 465.9 versus 592.5 µm (branch length, P=0.01), 22.5 versus 29.0 (maximum branch order, P=0.001), and 165.3 versus 311.7 (number of terminations, P=0.008). Furthermore, the dendritic spine density was reduced in essential tremor cases (medians=0.82 versus 1.02 µm(-1), P=0.03). Our demonstration of regressive changes in Purkinje cell dendritic architecture and spines in essential tremor relative to control brains provides additional evidence of a pervasive abnormality of Purkinje cell biology in this disease, which affects multiple neuronal cellular compartments including their axon, cell body, dendrites and spines.

Essential Tremor Followed by Progressive Supranuclear Palsy: Postmortem Reports of 11 Patients

Abstract For many years, clinicians have commented on the development ofsigns of parkinsonism among their essential tremor (ET) patients, but the links between ET and parkinsonism are not well understood. We report 11 (12.4%) of 89 ET patients who were prospectively collected at the Essential Tremor Centralized Brain Repository during the course of its first 9 years. All patients had long-standing ET (median duration, 38 years); there was a 5- to 49-year latency from the onset of ET to the development of either parkinsonism or dementia.Despite the presence of parkinsonism or dementia during life, none had been diagnosed clinically with progressive supranuclear palsy(PSP). All 11 received the postmortem diagnosis of PSP. The prevalence of PSP in this ET sample (12.4%) is clearly larger than the population prevalence of PSP (0.001%–0.0065%). Itis also 2 to 5 times the proportion of normal cases with incidental PSP in 2 previous autopsy series. This case series raises the questions of an association between ET and PSP, whether ET patients are at anincreased risk of developing PSP, and what the proportion of ETpatients who develop presumed Parkinson disease or Alzheimer disease in life actually have PSP (i.e. ET + PSP).

Cerebellar Pathology of a Dual Clinical Diagnosis: Patients with Essential Tremor and Dystonia

Background Clinical studies have implicated the cerebellum in the pathogenesis of essential tremor (ET), and recent postmortem studies have identified structural changes in the ET cerebellum. While the basal ganglia have traditionally been implicated in dystonia, cerebellar involvement has been suggested as well, and a recent study showed Purkinje cell (PC) loss. We conducted a detailed postmortem examination of the brain in four individuals with clinical diagnoses of ET and dystonia, and hypothesized that pathological changes in the cerebellum would be greater in these four ET cases than in published ET cases without dystonia. Methods After a complete neuropathological assessment, a standard parasagittal neocerebellar tissue block was harvested in each brain. One 7‐µm thick section was stained with luxol fast blue/hematoxylin and eosin, and one section with the Bielschowsky method. We quantified PCs, torpedoes, heterotopic PCs, PC dendritic swellings, and basket cell changes. Results Two ET+dystonia cases had more microscopic changes in the cerebellum than published ET cases; the other two cases had similar changes to published ET cases. Discussion This is the first report that uses human autopsy tissue to study patients with both ET and dystonia. The findings were heterogeneous. Additional studies, with larger samples, are needed.

Neurofilament protein levels: quantitative analysis in essential tremor cerebellar cortex.

Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and α-internexin in cerebellar tissue from 47 ET cases versus 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or α-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28±0.47 vs. 1.06±0.32, p=0.02; and 1.38±0.75 vs. 1.00±0.42, p=0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET.

Macroautophagy Abnormality in Essential Tremor

Macroautophagy is a cellular mechanism for the clearance of protein aggregates and damaged organelles. Impaired macroautophagy has been observed in neurodegenerative disorders. We investigated the macroautophagy pathway in essential tremor (ET) cases compared to age-matched controls. We analyzed microtubule-associated protein light chain 3-II (LC3-II), S6K, phosphorylated S6K, beclin-1, and mitochondrial membrane proteins levels by Western blot in the post-mortem cerebellum of 10 ET cases and 11 controls. We also performed immunohistochemistry in 12 ET cases and 13 controls to quantify LC3 clustering in Purkinje cells (PCs). LC3-II protein levels were significantly lower in ET cases vs. controls on Western blot (0.84±0.14 vs. 1.00±0.14, p = 0.02), and LC3-II clustering in PCs by immunohistochemistry was significantly lower in ET cases vs. controls (2.03±3.45 vs. 8.80±9.81, p = 0.03). In ET cases, disease duration was inversely correlated with LC3-II protein level (r = −0.64, p = 0.046). We found that mitochondrial membrane proteins were accumulated in ET (TIM23: 1.36±0.11 in ET cases vs. 1.00±0.08 in controls, p = 0.02; TOMM20: 1.63±0.87 in ET cases vs. 1.00±0.14 in controls, p = 0.03). Beclin-1, which is involved in macroautophagy, was strikingly deficient in ET (0.42±0.13 vs. 1.00±0.35, p<0.001). Decreased macroautophagy was observed in the ET cerebellum, and this could be due to a decrease in beclin-1 levels, which subsequently lead to mitochondrial accumulation as a result of autophagic failure. This provides a possible means by which perturbed macroautophagy could contribute to PC pathology in ET.

Parallel fiber counts and parallel fiber integrated density are similar in essential tremor cases and controls

Essential tremor (ET) is one of the most common neurological diseases, yet the number of published postmortem examinations is limited, and the range of pathological changes associated with this disease has yet to be fully catalogued. Aside from Purkinje cell (PC) loss and increased numbers of PC axonal swellings (torpedoes) in ET, hypertrophic changes in basket cell axonal plexuses have recently been identified [6], thus triggering the question whether other compensatory architectonic changes occur in the molecular or PC layers in this disease. To our knowledge, parallel fibers (PFs) have not been systematically quantified in ET cases versus matched controls. Postmortem cerebellar tissue was obtained from 20 ET cases without Lewy bodies and 19 non-diseased age-matched controls at the Essential Tremor Centralized Brain Repository (ETCBR), Columbia University, New York. All brains had complete neuropathological assessment [4]. Ascertainment of ET cases and controls and diagnosis of ET cases has been described [4]. A standard 3 × 20 × 25 mm parasagittal neocerebellar cortex tissue block was harvested from each brain. Paraffin sections (7 µm thick) were stained with a modified Bielschowsky silver method. Each brain had standardized measurement of brain weight (grams), postmortem interval (PMI), neurofibrillary pathology staging of Braak et al., and Consortium to Establish a Registry for AD (CERAD) rating [1, 7]. PCs and torpedoes were quantified as described [4]. Digital images of the Bielschowsky neocerebellum preparations were obtained from two selected regions in each brain using a Zeiss Axioplan 2 microscope fit with an Axiocam HR digital camera (20× objective lens). The two selected regions, both from the most centrally located folium in the section, were a distal gyrus and a distal sulcus. Each selected region was 200 µm in width and situated just above the PC layer (Fig. 1a, b). The height of each region, 160 µm, allowed for quantification of most of the stained PFs in the molecular layer. Each PF was manually traced using the tracing tool in Adobe Photoshop Element 6.0 (Fig. 1c). PFs were identified as the fibers oriented parallel to the PC layer. Fibers oriented >45 degrees to the PC layer were excluded, as were PC dendrites and baskets. Images of the PF tracings were inverted and imported into Image J (NIH), where integrated density was calculated, as described [4]. We averaged five randomly-selected background values (high in the molecular layer, with no visible PFs) and divided the integrated density by the average background value to obtain a PF integrated density (PFID) for each of the two regions. As the PFIDs in the two regions were highly correlated (Pearson’s r = 0.66, p < 0.001), we reported the average of those two PFIDs. Also, a PF count (PFC) was obtained by drawing an index line at the center of each image, and counting the number of PFs that crossed that line (Fig. 1b). The PFC we report was the average of those two PF counts, and was highly correlated with the PFID (Pearson’s r = 0.59, p < 0.001). Fig. 1 a Cerebellar cortical section (200× magnification, modified Bielschowsky silver method), showing PFs. A selected region was 200 µm in width and situated just above the PC layer. PFs within that region were analyzed. b A PFC was obtained ... Cases and controls were similar in age, gender, brain weight and PMI but, as expected, cases had more torpedoes and lower PC counts than controls (Table 1). Braak AD scores were higher in ET cases than controls, although CERAD scores did not differ (Table 1). The PFID and PFC did not correlate with age, brain weight, PMI, torpedoes, Braak AD stage, or CERAD score. The PFC correlated weakly and inversely with PC counts (Pearson’s r = −0.34, p = 0.04) and was lower in women than men (p = 0.005). Cases and controls were similar with respect to both the PFID and the PFC (Table 1). We assessed the role of several possible confounding factors. In a linear regression analysis that adjusted for gender, Braak AD stage and PC counts, PFC (outcome variable) did not differ by case–control status (beta = 1.54, p = 0.37). In a linear regression analysis that adjusted for Braak AD stage, PFID did not differ by case–control status (beta = 2899.4, p = 0.80). Table 1 Clinical and pathological features of ET cases and controls One limitation of this study is that the Bielschowsky method may not stain all PFs, yet we have no reason to suspect that this would be differential between cases and controls. Also, some of the fibers oriented parallel to the PC layer may have been basket cell processes rather than PCs. Finally, use of other staining methods (e.g., neurofilament antibody) could allow for additional case–control comparisons. Our analyses did not indicate a reduction in PFs in ET. Studies in PC and granule cell death demonstrate that different pathways are involved. PC death involves an autophagic pathway whereas granule cell death is predominantly apoptotic [2, 3]. In sporadic Creutzfeldt-Jakob disease, another cerebellar degenerative disease, granule cell death is marked yet PCs are relatively preserved [5], providing an example of the dissociation between PC and granule cell death. Our findings suggest that in ET, cerebellar degeneration is specific to PCs whereas PFs may be preserved.