Michelle Lee

Purkinje cell axonal anatomy: quantifying morphometric changes in essential tremor versus control brains.

Growing clinical, neuro-imaging and post-mortem data have implicated the cerebellum as playing an important role in the pathogenesis of essential tremor. Aside from a modest reduction of Purkinje cells in some post-mortem studies, Purkinje cell axonal swellings (torpedoes) are present to a greater degree in essential tremor cases than controls. Yet a detailed study of more subtle morphometric changes in the Purkinje cell axonal compartment has not been undertaken. We performed a detailed morphological analysis of the Purkinje cell axonal compartment in 49 essential tremor and 39 control brains, using calbindin D28k immunohistochemistry on 100-µm cerebellar cortical vibratome tissue sections. Changes in axonal shape [thickened axonal profiles (P = 0.006), torpedoes (P = 0.038)] and changes in axonal connectivity [axonal recurrent collaterals (P < 0.001), axonal branching (P < 0.001), terminal axonal sprouting (P < 0.001)] were all present to an increased degree in essential tremor cases versus controls. The changes in shape and connectivity were significantly correlated [e.g. correlation between thickened axonal profiles and recurrent collaterals (r = 0.405, P < 0.001)] and were correlated with tremor duration among essential tremor cases with age of onset >40 years. In essential tremor cases, thickened axonal profiles, axonal recurrent collaterals and branched axons were 3- to 5-fold more frequently seen on the axons of Purkinje cells with torpedoes versus Purkinje cells without torpedoes. We document a range of changes in the Purkinje cell axonal compartment in essential tremor. Several of these are likely to be compensatory changes in response to Purkinje cell injury, thus illustrating an important feature of Purkinje cells, which is that they are relatively resistant to damage and capable of mobilizing a broad range of axonal responses to injury. The extent to which this plasticity of the Purkinje cell axon is partially neuroprotective or ultimately ineffective at slowing further cellular changes and cell death deserves further study in essential tremor.

Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression

Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with ICV infusion of the specific MC3-R agonist, [D-Trp(8)]-gamma-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that ICV infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake.

Review of physiology, clinical manifestations, and management of hypothalamic obesity in humans

Hypothalamic injury from acquired structural damage due to infiltrative disease, tumor, or their treatment aftereffects frequently results in the development of an obesity syndrome characterized by a rapid, unrelenting weight gain that may be accompanied by severe hyperphagia. Weight gain occurs from the disruption of the normal homeostatic functioning of the hypothalamic centers responsible for controlling satiety and hunger and regulating energy balance with resulting hyperphagia, autonomic imbalance, reduction of energy expenditure, and hyperinsulinemia. Curtailment of weight increase has traditionally been refractory to usual dietary and lifestyle interventions. Pharmacotherapy targeting insulin secretion and augmenting sympathetic output have been attempted to promote weight loss or attenuate weight gain. In addition, case reports suggest that bariatric surgery may be an effective treatment option for these patients. Hormonal deficits are often present, and their management may also have consequences for weight control. Hypothalamic obesity confers significant morbidity and mortality, and there is a need for greater elucidation of its risk factors and pathogenesis so that more effective interventions can be developed.

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.

Quantification of cerebellar hemispheric purkinje cell linear density: 32 ET cases versus 16 controls.

Although essential tremor (ET) is among the most prevalent neurological diseases, its precise pathogenesis is not understood. Purkinje cell loss has been observed in some studies and is the focus of interest and debate. Expressing these data as Purkinje cells/layer length allows one to adjust for the inherent curved nature of the cerebellar folia. Capitalizing on the Essential Tremor Centralized Brain Repository, we quantified Purkinje cell linear density in cases versus controls. Free‐floating 100‐μm parasagittal cerebellar hemispheric sections were subjected to rabbit polyclonal anti‐Calbindin D28k antibody, and 10 random fields/brain were selected for quantification of Purkinje cells/mm−1 Purkinje cell layer. Purkinje cell linear density was lower in 32 ET cases than in16 controls (1.14u2009±u20090.32 vs. 1.35u2009±u20090.31/mm−1, Pu2009=u20090.03). Purkinje cell linear density was inversely associated with torpedo count (ru2009=u2009−0.38, Pu2009=u20090.028). The current sample of ET cases demonstrates a reduction in Purkinje cell number relative to that of controls. Greater Purkinje cell axonal remodeling (torpedoes) was found in individuals who had the most Purkinje cell drop out. The role of Purkinje cell loss in the pathogenesis of this disorder merits additional study.

Decreased EAAT2 protein expression in the essential tremor cerebellar cortex

Genetic polymorphisms in Solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SLC1A2) have been linked with essential tremor. SLC1A2 encodes excitatory amino acid transporter type 2 (EAAT2), which clears glutamate from the synaptic cleft. One postulated mechanism for essential tremor is the over-excitation of glutamatergic olivo-cerebellar climbing fibers, leading to excitotoxic death of Purkinje cells. Other glutamatergic excitatory signals are transmitted to Purkinje cells via parallel fibers of cerebellar granule neurons. Therefore, the expression level of glutamate transporters could be important in essential tremor pathogenesis. Using Western blotting, we compared the expression levels of the two main glutamate transporters in the cerebellar cortex, EAAT1 and EAAT2, in postmortem tissue from 16 essential tremor cases and 13 age-matched controls. We also studied the localization of EAAT1 and EAAT2 using immunohistochemistry in 10 essential tremor cases and 12 controls. EAAT1 protein levels were similar in cases and controls (1.12 ± 0.83 vs. 1.01 ± 0.69, p =0.71) whereas EAAT2 protein levels in essential tremor cases were only 1/3 of that in controls (0.35 ± 0.23 vs. 1.00 ± 0.62, p < 0.01). Interestingly, EAAT2, but not EAAT1, was expressed in astrocytic processes surrounding the Purkinje cell axon initial segment, a region of previously observed pathological changes in essential tremor. Our main finding, a significant reduction in cerebellar cortical EAAT2 protein levels in essential tremor, suggests that Purkinje cells in essential tremor might be more vulnerable to excitotoxic damage than those of controls.

Matching Asymmetry of Tremor with Asymmetry of Postmortem Cerebellar Hemispheric Changes in Essential Tremor

Although the number of postmortem studies in essential tremor (ET) has grown in recent years, clinical-pathological correlations remain limited. We are unaware of a study that has assessed whether the pathological changes in ET, if asymmetric, lateralize to the cerebellar hemisphere that is ipsilateral to the arm with more severe action tremor, as one would predict if the lesions were tremor producing. We compared postmortem changes in the right vs. left cerebellar hemispheres in ET and examined how these correlated with asymmetry of tremor on neurological examination. Action tremor in each arm was quantified using a reliable and valid clinical rating scale. Cases were divided into three clinical groups: tremor more severe on right, tremor more severe on left, and tremor symmetric. Calbindin D28k immunohistochemistry was performed on 100xa0μm vibrotome sections from a standard tissue block of both right and left neocerebellums to quantify Purkinje cell linear density, torpedo counts, and a group of previously described changes in Purkinje cell axonal shape (thickened axonal profiles) and connectivity (axon recurrent collaterals, axonal branching, terminal axonal sprouting, arciform axons, extent of recurrent collateral plexus). ET cases were divided into three postmortem groups: findings greatest on right, findings greatest on left, and findings symmetric. In 18 (72.0xa0%) of 25 ET cases, clinical and pathological features were concordant (i.e., both clinically and pathologically right-predominant (one case), both clinically and pathologically left-predominant (five cases), or both clinically and pathologically symmetric (12 cases), pu2009=u20090.007). In the remaining seven (28.0xa0%) ET cases, clinical and pathological data were not concordant, and in none were they completely discordant (i.e., tremor was more severe on the right, and postmortem cerebellar changes were paradoxically more severe on the left or vice versa). Among the seven ET cases with >20xa0% side-to-side difference in tremor severity, six cases (85.7xa0%) had the expected pathological asymmetry, with quantified postmortem cerebellar changes more marked ipsilateral to the more clinically affected side. We also created continuous measures of asymmetry. For the entire sample, there was a positive correlation between the clinical asymmetry index and the pathological asymmetry indexu2009=u20090.52, pu2009=u20090.01 (i.e., the right-left difference in clinical asymmetry was correlated with the right-left difference in postmortem changes). For the seven ET cases with clear clinical asymmetry, the correlation was even more robust (ru2009=u20090.78, pu2009=u20090.039). Clinical-pathological correlations are important in terms of understanding the significance of observed pathological changes. The correlation between clinical laterality or symmetry of tremor and pathological changes in the majority of ET cases provides additional evidence that the pathological changes in the cerebellum in ET are of patho-mechanistic importance.

Alzheimer's-related changes in non-demented essential tremor patients vs. controls: Links between tau and tremor?

BACKGROUNDnIn addition to tremor, patients with essential tremor (ET) may exhibit non-motor features, including a range of cognitive deficits. Several prospective, population-based epidemiological studies have reported an association between ET and incident dementia, especially Alzheimers disease (AD). Moreover, in a brain repository-based study, a larger than expected proportion of ET patients also developed pathological changes characteristic of progressive supranuclear palsy, further suggesting a link between ET and tau pathology.nnnMETHODSnWe selected a group of ET patients that were free of dementia clinically and without AD on postmortem examination. Our hypothesis was that neuronal tauopathic burden would be higher in the brains of these ET patients compared to controls. We compared Braak stage for neuronal tangles and Consortium to Establish a Registry for Alzheimers Disease (CERAD) scores for neuritic plaques in the two groups.nnnRESULTSnThe two groups were similar in age (82.6xa0±xa06.0 vs. 80.4xa0±xa08.1, pxa0=xa00.22). The 40 ET patients had a higher Braak neurofibrillary stage than 32 controls (means: 2.2xa0±xa01.2 vs. 1.2xa0±xa01.1; medians: 2.0 vs. 1.0, pxa0<xa00.001). Meanwhile, CERAD scores for neuritic plaques were similar in patients and controls (means: 0.6xa0±xa00.9 vs. 0.5xa0±xa00.6; medians: 0.0 vs. 0.0, pxa0=xa00.83).nnnCONCLUSIONnWhile ET itself is not a tauopathy (i.e., a neurodegenerative disorder among whose main features are accumulation of hyperphosphorylated tau protein), ET may predispose individuals to accumulate more widespread cellular tau aggregates, and thus tau could play a central role in the cognitive impairment that can accompany ET.

Cellular density in the cerebellar molecular layer in essential tremor, spinocerebellar ataxia, and controls.

BACKGROUNDnIt would be useful to identify additional postmortem markers of Purkinje cell loss in essential tremor (ET). In hereditary cerebellar ataxia, Purkinje cell loss has been reported to result in a secondary increase in the density of the remaining cell populations in the cerebellar molecular layer. However, this phenomenon has not been studied in ET. We quantified cerebellar molecular layer cellular density in 15xa0ET cases, 15 controls, and 7 spinocerebellar ataxia (SCA) cases (2:2:1 ratio).nnnMETHODSnA standard neocerebellar tissue block was stained with Luxol fast blue Hematoxylin & Eosin. Within 5 selected fields, cell soma (e.g., stellate, basket, and glial cell bodies) were counted. Cellular density was the number of cells/cm(2).nnnRESULTSnThe Purkinje cell count differed across the three groups (pxa0<xa00.001), with the highest counts in controls, intermediate counts in ET cases and lowest counts in SCA cases. ET cases and controls had similar molecular layer cellular density (pxa0=xa00.79) but SCA cases had higher values than both groups (pxa0<xa00.01). A robust inverse correlation between Purkinje cell count and molecular layer cellular density (i.e., brains with more Purkinje cell loss had higher molecular layer cellular density), observed in SCA and controls (rxa0=xa0-0.55, pxa0=xa00.008), was not observed in ET cases.nnnDISCUSSIONnAlthough Purkinje cell counts were reduced in ET cases compared to controls, an increase in molecular layer cellular density was not evident in ET. The increase in molecular layer cellular density, observed in SCA cases, may require a more marked loss of PCs than occurs in ET.

Excitatory Amino acid transporter expression in the essential tremor dentate nucleus and cerebellar cortex: A postmortem study

BACKGROUNDnGenome-wide association studies have revealed a link between essential tremor (ET) and the gene SLC1A2, which encodes excitatory amino acid transporter type 2 (EAAT2). We explored EAAT biology in ET by quantifying EAAT2 and EAAT1 levels in the cerebellar dentate nucleus, and expanded our prior analysis of EAAT2 levels in the cerebellar cortex.nnnOBJECTIVEnTo quantify EAAT2 and EAAT1 levels in the cerebellar dentate nucleus and cerebellar cortex of ET cases vs.nnnCONTROLSnnnnMETHODSnWe used immunohistochemistry to quantify EAAT2 and EAAT1 levels in the dentate nucleus of a discovery cohort of 16xa0ET cases and 16 controls. Furthermore, we quantified EAAT2 levels in the dentate nucleus in a replicate cohort (61xa0ET cases, 25 controls). Cortical EAAT2 levels in all 77xa0ET cases and 41 controls were quantified.nnnRESULTSnIn the discovery cohort, dentate EAAT2 levels were 1.5-fold higher in 16xa0ET cases vs. 16 controls (pxa0=xa00.007), but EAAT1 levels did not differ significantly (pxa0=xa00.279). Dentate EAAT2 levels were 1.3-fold higher in 61xa0ET cases vs. 25 controls in the replicate cohort (pxa0=xa00.022). Cerebellar cortical EAAT2 levels were 20% and 40% lower in ET cases vs. controls in the discovery and the replicate cohorts (respective p valuesxa0=xa00.045 andxa0<xa00.001).nnnCONCLUSIONnEAAT2 expression is enhanced in the ET dentate nucleus, in contrast to differentially reduced EAAT2 levels in the ET cerebellar cortex, which might reflect a compensatory mechanism to maintain excitation-inhibition balance in cerebellar nuclei.