Despite its being one of the most commonly observed neurological disorders, neuropathological studies of essential tremor (ET) are rare. There have been surprisingly few autopsy studies and even fewer case-control comparisons. The primary objective was to describe and quantify the pathological changes in 33 ET and 21 control brains. A secondary objective was to correlate clinical and pathological features. We examined autopsy tissue from the Essential Tremor Centralized Brain Repository. Eight (24.2%) of the 33 ET brains had Lewy bodies in the brainstem, mainly in the locus ceruleus. However, the majority of ET brains (25/33, 75.8%) had no Lewy bodies, but had pathological changes in the cerebellum. The mean number of Purkinje cells per 100x field was reduced in ET cases without Lewy bodies (6.6 +/- 2.4 versus 9.6 +/- 3.4, P < 0.01), and there were approximately 7x more Purkinje cell torpedoes per section (12.6 +/- 7.9 versus 1.7 +/- 1.4, P < 0.001) compared to controls. ET cases without Lewy bodies also had degeneration of the dentate nucleus (two cases). Other findings in ET cases were Purkinje cell heterotopias and dendrite swellings. Lewy body ET cases were older than ET cases without Lewy bodies. Several trends were observed in ET cases without Lewy bodies, including a younger age of onset of tremor and higher proportions with gait difficulty and family history of ET. The pathological changes of ET seem to be heterogeneous and degenerative. The majority have cerebellar changes without Lewy bodies; a smaller proportion has brainstem Lewy bodies. The clinical differences between cases with versus without Lewy bodies require additional study.

Many patients with SARS-CoV-2 infection develop neurological signs and symptoms, though, to date, little evidence exists that primary infection of the brain is a significant contributing factor. We present the clinical, neuropathological, and molecular findings of 41 consecutive patients with SARS-CoV-2 infections who died and underwent autopsy in our medical center. The mean age was 74 years (38–97 years), 27 patients (66%) were male and 34 (83%) were of Hispanic/Latinx ethnicity. Twenty-four patients (59%) were admitted to the intensive care unit (ICU). Hospital-associated complications were common, including 8 (20%) with deep vein thrombosis/pulmonary embolism (DVT/PE), 7 (17%) patients with acute kidney injury requiring dialysis, and 10 (24%) with positive blood cultures during admission. Eight (20%) patients died within 24 hours of hospital admission, while 11 (27%) died more than 4 weeks after hospital admission. Neuropathological examination of 20–30 areas from each brain revealed hypoxic/ischemic changes in all brains, both global and focal; large and small infarcts, many of which appeared hemorrhagic; and microglial activation with microglial nodules accompanied by neuronophagia, most prominently in the brainstem. We observed sparse T lymphocyte accumulation in either perivascular regions or in the brain parenchyma. Many brains contained atherosclerosis of large arteries and arteriolosclerosis, though none had evidence of vasculitis. Eighteen (44%) contained pathologies of neurodegenerative diseases, not unexpected given the age range of our patients. We examined multiple fresh frozen and fixed tissues from 28 brains for the presence of viral RNA and protein, using quantitative reverse-transcriptase PCR (qRT-PCR), RNAscope, and immunocytochemistry with primers, probes, and antibodies directed against the spike and nucleocapsid regions. qRT-PCR revealed low to very low, but detectable, viral RNA levels in the majority of brains, although they were far lower than those in nasal epithelia. RNAscope and immunocytochemistry failed to detect viral RNA or protein in brains. Our findings indicate that the levels of detectable virus in COVID-19 brains are very low and do not correlate with the histopathological alterations. These findings suggest that microglial activation, microglial nodules and neuronophagia, observed in the majority of brains, do not result from direct viral infection of brain parenchyma, but rather likely from systemic inflammation, perhaps with synergistic contribution from hypoxia/ischemia. Further studies are needed to define whether these pathologies, if present in patients who survive COVID-19, might contribute to chronic neurological problems.

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signaling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by PEX19 and PEX5, respectively, and peroxisome-localized TSC functioned as a Rheb GAP to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, abrogated peroxisome localization, Rheb GAP activity, and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS and peroxisome-localization deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for TSC in Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript responding to ROS at the peroxisome, and identify the peroxisome as a signaling organelle involved in regulation of mTORC1.Tuberous sclerosis complex (TSC) is a hereditary hamartoma syndrome caused by defects in either the TSC1 or TSC2 genes 1, 2 . The TSC tumor suppressor is a heterodimer comprised of tuberin (TSC2), a GTPase activating protein (GAP), and its activation partner hamartin (TSC1), which localizes the TSC tumor suppressor to endomembranes and protects TSC2 from proteasomal degradation 3,4 . TSC inhibits the activity of the small GTPase Rheb to repress mammalian target of rapamycin complex 1 (mTORC1) signaling, a negative regulator of autophagy [5][6][7][8][9][10][11][12] . mTORC1 is regulated by a variety of cellular stimuli including amino acids, mitogens such as insulin, glucose, and energy stress [13][14][15] . In the case of amino acids, which do not signal through TSC-Rheb pathway 15 , mTORC1 activity is regulated by the Rag GTPases, which form the Ragulator complex that localizes mTORC1 to the late endosome or lysosome compartment of cells [13][14][15][16][17][18] . We recently reported that TSC functions in a signaling node downstream of ataxia telangiectasia mutated (ATM) to repress mTORC1 in response to reactive oxygen species (ROS) 19 . However, identification of the specific subcellular compartment(s) in which the TSC tumor suppressor functions to regulate mTORC1 in response to ROS has heretofore remained elusive.Peroxisomes, carry out key metabolic functions in the cell including β-oxidation of fatty acids, and are a major source of cellular ROS 20,21 . Like mitochondria, peroxisomes are autonomously replicating organelles. Peroxisome biogenesis requires peroxin (PEX) proteins, which are essential for assembly of functional peroxisomes 22 . Specific PEX pro...

Background: Clinical and functional imaging evidence suggests that cerebellar dysfunction occurs in essential tremor (ET). In recent postmortem studies, we documented increased numbers of torpedoes (Purkinje cell axonal swellings) in ET patients without Lewy bodies. Purkinje cell loss, however, has never been rigorously assessed. Objective: To quantitatively assess the number of Purkinje cells in brains of ET patients and similarly aged controls. Methods: Postmortem cerebellar tissue was available in 14 ET cases (6 with Lewy bodies and 8 without Lewy bodies) and 11 controls. Calbindin immunohistochemistry was performed on paraffin sections of the cerebellum. Images were digitally recorded and blinded measurements of the number of Purkinje cells per millimeter of cell layer (linear density) were made. Results: Purkinje cell linear density was inversely correlated with age (r=−0.53, P=.006) and number of torpedoes (r=−0.42, P=.04). Purkinje cell linear density differed by diagnosis (mean [SD], controls, 3.46 [1.27] cells/ mm; ET cases with Lewy bodies, 3.33 [1.06] cells/mm; and ET cases without Lewy bodies, 2.14 [0.82] cells/ mm; P = .04), with the most significant difference between ET cases without Lewy bodies and controls, where the reduction was 38.2% (P=.04). In an adjusted linear regression analysis that compared ET cases without Lewy bodies with controls, decreased linear density (outcome variable) was associated with ET (␤=.56, P=.03). Conclusions: We demonstrated a reduction in Purkinje cell number in the brains of patients with ET who do not have Lewy bodies. These data further support the view that the cerebellum is anatomically, as well as functionally, abnormal in these ET cases.

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.

Peroxisomes play a central role in lipid metabolism, and their function depends on molecular oxygen. Low oxygen tension or von Hippel-Lindau (Vhl) tumor suppressor loss is known to stabilize hypoxia-inducible factors alpha (Hif-1α and Hif-2α) to mediate adaptive responses, but it remains unknown if peroxisome homeostasis and metabolism are interconnected with Hif-α signaling. By studying liver-specific Vhl, Vhl/Hif1α, and Vhl/Hif2α knockout mice, we demonstrate a regulatory function of Hif-2α signaling on peroxisomes. Hif-2α activation augments peroxisome turnover by selective autophagy (pexophagy) and thereby changes lipid composition reminiscent of peroxisomal disorders. The autophagy receptor Nbr1 localizes to peroxisomes and is likewise degraded by Hif-2α-mediated pexophagy. Furthermore, we demonstrate that peroxisome abundance is reduced in VHL-deficient human clear cell renal cell carcinomas with high HIF-2α levels. These results establish Hif-2α as a negative regulator of peroxisome abundance and metabolism and suggest a mechanism by which cells attune peroxisomal function with oxygen availability.

An 1110-base-pair cDNA clone for human cathepsin D was obtained by screening a XgtlO human hepatoma G2 cDNA library with a human renin exon 3 genomic fragment. Poly(A)+ RNA blot analysis with this cathepsin D clone demonstrated a message length of about 2.2 kilobases. The partial clone was used to screen a size-selected human kidney cDNA library, from which two cathepsin D recombinant plasmids with inserts of about 2200 and 2150 base pairs were obtained. The nucleotide sequences of these clones and of the XgtlO clone were determined. The amino acid sequence predicted from the cDNA sequence shows that human cathepsin D consists of 412 amino acids with 20 and 44 amino acids in a pre-and a prosegment, respectively. The mature protein region shows 87% amino acid identity with porcine cathepsin D but differs in having nine additional amino acids. Two of these are at the COOH terminus; the other seven are positioned between the previously determined junction for the light and heavy chains of porcine cathepsin D. A high degree of sequence homology was observed between human cathepsin D and other aspartyl proteases, suggesting a conservation of three-dimensional structure in this family of proteins.Cathepsin D is a lysosomal endoprotease that is present in all mammalian cells (for review, see ref. 1). It is a member of the aspartyl protease family, among which are the well-studied secretory enzymes renin, pepsin, and chymosin (2). Cathepsin D is the only aspartyl protease known to be lysosomal rather than secretory. Recently, the complete amino acid sequence for the mature protein of porcine cathepsin D has been determined (3). Alignment of this sequence with that of renin showed an overall 48.9o homology (3). In addition, the region surrounding the first activesite aspartyl (residue 32 in the pepsin numbering convention) was even more highly conserved.The entire human renin gene has now been cloned and its organization has been determined (4, 5 A human kidney cDNA library was screened for a fulllength clone. Total RNA was isolated by the guanidinium thiocyanate method (7) from a portion of surgically removed, perfused kidney and twice selected by chromatography on an oligo(dT)-cellulose column (8). Poly(A)+ RNA (29 ,ug) was reverse-transcribed under the conditions of Retzell et al. (9). The first-strand heteroduplex was converted to fully doublestranded cDNA by replacement synthesis (10). The doublestranded cDNA was then resolved by preparative electrophoresis in an agarose gel and cDNAs 1.8-2.5 kilobases (kb) long were cut out and isolated (11). These cDNAs were then oligo(dC)-tailed (12) and annealed with plasmid pUC19 (13) that had been oligo(dG)-tailed at the Sac I site. E. coli K-12 strain DH-1 (14) was transformed and filter replicas of ampicillin-resistant colonies were prepared by the method of Hanahan and Meselson (15 Filters were washed at 45°C in 0.1 x NaCl/Cit/0.1% NaDodSO4 prior to autoradiography. Positive phage were plaque-purified (11) at least three times.Abbreviations: kb, kilobase(s); bp, bas...

Many patients with SARS-CoV-2 infection develop neurological signs and symptoms, though, to date, little evidence exists that primary infection of the brain is a significant contributing factor. We present the clinical, neuropathological, and molecular findings of 41 consecutive patients with SARS-CoV-2 infections who died and underwent autopsy in our medical center. The mean age was 74 years (38-97 years), 27 patients (66%) were male and 34 (83%) were of Hispanic/Latinx ethnicity. Twenty-four patients (59%) were admitted to the intensive care unit (ICU). Hospital-associated complications were common, including 8 (20%) with deep vein thrombosis/pulmonary embolism (DVT/PE), 7 (17%) patients with acute kidney injury requiring dialysis, and 10 (24%) with positive blood cultures during admission. Eight (20%) patients died within 24 hours of hospital admission, while 11 (27%) died more than 4 weeks after hospital admission. Neuropathological examination of 20-30 areas from each brain revealed hypoxic/ischemic changes in all brains, both global and focal; large and small infarcts, many of which appeared hemorrhagic; and microglial activation with microglial nodules accompanied by neuronophagia, most prominently in the brainstem. We observed sparse T lymphocyte accumulation in either perivascular regions or in the brain parenchyma. Many brains contained atherosclerosis of large arteries and arteriolosclerosis, though none had evidence of vasculitis. Eighteen (44%) contained pathologies of neurodegenerative diseases, not unexpected given the age range of our patients. We examined multiple fresh frozen and fixed tissues from 28 brains for the presence of viral RNA and protein, using quantitative reverse-transcriptase PCR (qRT- PCR), RNAscope, and immunocytochemistry with primers, probes, and antibodies directed against the spike and nucleocapsid regions. qRT-PCR revealed low to very low, but detectable, viral RNA levels in the majority of brains, although they were far lower than those in nasal epithelia. RNAscope and immunocytochemistry failed to detect viral RNA or protein in brains. Our findings indicate that the levels of detectable virus in COVID-19 brains are very low and do not correlate with the histopathological alterations. These findings suggest that microglial activation, microglial nodules and neuronophagia, observed in the majority of brains, do not result from direct viral infection of brain parenchyma, but rather likely from systemic inflammation, perhaps with synergistic contribution from hypoxia/ischemia. Further studies are needed to define whether these pathologies, if present in patients who survive COVID-19, might contribute to chronic neurological problems.