Principal Investigator:
Holly Shill, MD, FAAN, Barrow Neurological Institute, Phoenix, AZ
Co-Investigator:
Malvindar Singh-Bains, PhD, Centre for Brain Research, University of Auckland, New Zealand
Consultant:
Thomas G. Beach, MD, PhD, Banner Sun Health Research Institute, Sun City, AZ
PROJECT SUMMARY
A significant barrier in essential tremor (ET) research and development of therapeutics has been the lack of good laboratory models to study ET. This is largely because there are no well-defined genetic changes nor clear changes seen within the brain itself which could be used to model ET in the lab. For the past 15 years, there have been a number of research publications exploring the neuropathology of ET. Neuropathology means examining changes that are seen in the number of brain cells and their ability to maintain connections, most often studied at the microscopic level. Due to the progressive nature of ET combined with findings of unsteadiness and incoordination in advancing ET, the cerebellum has been a logical place to look for evidence of neurodegeneration as this is the area of the brain responsible for these symptoms. Cerebellar involvement in ET is supported by human studies of the metabolism and electrical activity in the brain with both types of studies showing overactivity of the cerebellum. Several research groups have looked at counting of cerebellar Purkinje cells and other changes in the cerebellum with conflicting results. There is a need for additional studies to clarify cerebellar pathological findings in ET in order to support or refute the hypothesis that ET may be a neurodegenerative disorder of the cerebellum. This new study funded by the IETF will use enhanced methods to study findings in the human cerebellum. If the findings can be confirmed, it will open the potential to develop better models of ET as well as potential targets for treatment.
The goal of this project is to determine whether essential tremor is a neurodegenerative disease affecting the cerebellum.
Aim 1: The first aim of this project will be to quantify the number of Purkinje cells (PCs) in the cerebellum using brain tissue from autopsied ET patients with the analysis being done by an independent, blinded neuropathology group who are experts in conducting unbiased design-based stereological studies of cerebellar Purkinje cell numbers. Combining these estimates of Purkinje cell number with cerebellar volume (see our recent publication from the same subjects, we will, for the first time, be able to reliably estimate the total numbers of Purkinje cells in the cerebella from ET and control subjects. Cases of ET will be compared to both age and sex matched control populations, with the independent pathology group kept blinded to clinical information. Cases will be selected using current diagnostic criteria for ET and ET Plus. Cerebellar samples will be used from motor (neocerebellum) cerebellum with results adjusted for age and age-related pathologies. Free floating immunohistochemistry staining protocols will be carried out on the neocerebellum sections using standard single peroxidase labelling techniques as detailed previously. Random sections of the neocerebellum will be immunolabeled with the calcium-binding protein calbindin to specifically and reliably identify Purkinje neurons.
Aim 2: While Aim 1 focuses on design-based quantification of PCs in ET/ET-Plus versus neurologically normal control cases, the second aim will assess more subtle indicators of disease in the cerebellum including the somal volume of Purkinje cell bodies, molecular layer volume and the degree of PC process arborization (number of branches, length of processes) within the neocerebellum using high throughput measures.
Progress Reports
January 2025
In January 2024, work began to identify cases for selection for the project. A database search was done, identifying 250 cases with ET. These cases were all reviewed individually, blinded to any pathological data, to select cases that would be appropriate for further study. Out of 250 ET, we excluded 122 due to clinical dementia and/or parkinsonism. Another 62 were excluded due to shorter tremor duration/ low amplitude tremor or other clinical factors such as history of alcoholism. This left 66 cases. This was then sent to the pathology team who did further reviews to exclude those with concomitant pathology that would directly affect the cerebellum such as PSP, MSA and cerebellar infarcts. Out of 26 remaining cases, 15 were identified with adequate tissue for study. Once the ET cases were identified, the control population was chosen with identical exclusionary factors.
It took some time for the contracting and materials transfer agreements to be put in place to send tissue from the Banner Sun Health brain bank in Sun City, AZ to Dr. Bains-Singh at the Centre for Brain Research, University of Auckland, New Zealand. This occurred in the summer of 2024 and the tissue blocks were sent. The tissue work began in Auckland in September 2024. The first steps were in tissue processing and then serially sectioning the samples. To date, approximately 20% of cases have been cut. The stereology microscope is being updated and should be done at the end of January 2025. The immunohistochemistry work will then begin in February 2025. The tissue work will continue until the end of 2025. Once completed, the results will be returned to the PI for unblinding, statistical analysis and publication.
July 2025
In the fall of 2024, brain tissue from 28 individuals was sent from Banner Sun Health Research Institute in Sun City, Arizona, USA to the University of Auckland, New Zealand for analysis. This tissue was processed to make it appropriate for cutting and staining. Overall, 78 blocks from 28 cases were cut into very thin slices, which produced over 11,000 sections ready for experimentation. The process then shifted to mounting the cut tissue onto glass slides which is done very carefully to avoid tangling or folding the delicate tissue. The slides were then dried for 1-2 weeks before the staining process could commence. Once dry, the tissue is now ready for staining. The staining process is also a time intensive task that cannot be rushed.
To date, we have now stained 12 cases, which is approximately 132 sections. Once staining is complete, the analysis can then take place.
January 2026
Recap of the last report
In the previous report, all formalin-fixed post-mortem human cerebella hemispheres from 14 ET cases matched with 14 control cases were cut into serial sagittal sections at 70 µm thickness using a sliding microtome. For all cases, tissue blocks were cut from the neocerebellum (the most lateral component of the cerebellum to the medial start point of the deep cerebellar nuclei). The cut sections are stored in PBS-azide in 48-well plates at 4°C. We were in the process of cresyl violet staining all cases at a 1:40 sampling interval (every 40th section was stained). This staining was a lengthy process due to the importance of careful mounting of sections onto microscope slides and ensuring the sections dried entirely before microscope examination. The purpose of this staining was to determine:
I. Whether the sections from each case are suitable for stereological counting based on our neuroanatomical selection criteria.
II. A suitable sampling rate for stereology to ensure enough Purkinje cells are quantified.
III. The boundaries of the region of interest for stereology are captured consistently across all cases.
In addition, we conducted our first experimental run to determine the best antibody condition for immunohistochemical staining of Purkinje neurons using standard 3’3-diaminobenzidine (DAB) labelling.
Progress made since the last report
We have completed staining all essential tremor and control which was approximately 308 sections in total for neuroanatomical examination. We have gone through all the cases and have identified the region of interest counting. The technician is now trained to use the counting tool. We now are in the process of setting up the inclusion criteria for Purkinje cell counting based on settings determined in previous studies from our laboratory. A flow chart of the criteria is being created to ensure consistency and confidence that the objects of interest (Purkinje cells) are being counted. Counting will commence in February 2026 and we look forward to sharing preliminary counting data in our next report.
References:
Burnett, O. (2023). Characterising gliosis in the X-linked Dystonia Parkinsonism human striatum. [Unpublished Bachelor of Science (Honours) in Biomedical Science dissertation, University of Auckland].
Sehajpal, K. (2024). Substantia nigra in cases of X-linked Dystonia Parkinsonism. [Unpublished Master of Biomedical Science thesis, University of Auckland].
Sehji, T. (2015). Purkinje cell loss in the human cerebellum in Huntington’s disease. [Unpublished Bachelor of Science (Honours) in Biomedical Science dissertation, University of Auckland].
Singh-Bains, M. K., Mehrabi, N. F., Sehji, T., Austria, M. D. R., Tan, A. Y. S., Tippett, L. J., Dragunow, M., Waldvogel, H. J., & Faull, R. L. M. (2019a). Cerebellar degeneration correlates with motor symptoms in Huntington’s disease. Annals of neurology, 85(3), 396–405. https://doi.org/10.1002/ana.25413
Singh-Bains, M. K., Linke, V., Austria, M. D. R., Tan, A. Y. S., Scotter, E. L., Mehrabi, N. F., Faull, R. L. M., & Dragunow, M. (2019b). Altered microglia and neurovasculature in the Alzheimer’s disease cerebellum. Neurobiology of Disease, 132, 104589. https://doi.org/10.1016/j.nbd.2019.10458
July 2026
Progress made since the last report
The optimisation experiments found the antibody condition of 1:2500 for anti-calbindin was sufficient to stain all sections completely across the neocerebellar block, irrespective of control or disease condition, including the larger sections from the medial face of the tissue block, which were previously understained at 1:5000 (Figure 1). Using the higher antibody condition, all sections for all cases were immunostained using 3,3’-diaminobenzidine (DAB) and subjected to the manual mounting and dehydration process. To ensure the sampling of the tissue remains unbiased and random, a condition for design-based stereology, all sections that were selected for DAB immunostaining were selected by systematic random sampling (SRS), where every 40th section was taken after a random starting point. This is an unbiased and efficient method of selecting a representative sample of the tissue (Glaser et al, 2007; Mehrabi et al, 2018).
View the full update online.