Illuminating stomatal responses to red light: establishing the role of Ci-dependent vs -independent mechanisms in control of stomatal behaviour.

The stomatal response to red light appears to link stomatal conductance (gs) with photosynthetic rates. Initially, it was suggested that changes in intercellular CO2 (Ci) provide the main cue via a Ci-dependent response. However, evidence for Ci-independent mechanisms suggests an additional, more direct relationship with photosynthesis. While both Ci-dependent and -independent mechanisms clearly function in stomatal red-light responses, little is known regarding their relative contribution. The present study aimed to quantify the relative magnitude of Ci-dependent and -independent mechanisms on the stomatal red-light response, to characterise their interplay and to assess the putative link between plastoquinone (PQ) redox state and Ci-independent stomatal responses. Red light response curves measured at a range of Ci values for wild-type Arabidopsis thaliana (Col-0) and the CO2 hyposensitive mutant, ca1ca4, allowed deconvolution of Ci-dependent and -independent pathways. Surprisingly, we observed that both mechanisms contribute equally to stomatal red-light responses, but Ci-independent stomatal opening is suppressed at high Ci. The present data are also consistent with the involvement of PQ redox in coordinating the Ci-independent component. Overall, it seems that while Ci-independent mechanisms are distinct from responses to Ci, interplay between these two pathways is important to facilitate effective coordination between gs and photosynthesis.

Management & surveillance of rectal neuroendocrine tumours: a single-centre retrospective analysis.

BACKGROUND: Rectal neuroendocrine tumours (rNETs) are rare but are increasing in incidence. Current management and surveillance recommendations are based on low-grade evidence. Follow-up practices are often inconsistent and costly. This retrospective study analyses a single-centre's experience with rNETs to assess incidence, management practices, outcomes, and guideline adherence. METHODS: This is a single-centre retrospective study from Queensland Australia, spanning from 2012 to 2023. Twenty-eight rNET cases met inclusion criteria. Examined parameters included incidence, management, outcomes and adherence to European Neuroendocrine Tumour Society (ENETS) guidelines. R1 resection rate was analysed for associations with resection technique and lesion recognition and recurrence rate was assessed in all patients. RESULTS: This study shows an increasing incidence of rNETs during the study period, reflecting a global trend. R1 resection rate at initial endoscopy was 75%. There was a general lack of advanced endoscopic techniques utilized and poor lesion recognition, however a statistically significant correlation was not established between these factors and an R1 result (P < 0.05). Most patients with an R1 result had subsequent re-resection to render the result R0, however five patients (33%) underwent surveillance with no reports of recurrence on follow-up. Overall, follow-up practices in our cohort were inconsistent and did not adhere to guidelines. CONCLUSION: rNETs are increasing in incidence, emphasizing the need for standardized management and surveillance. Further training is required for rNET recognition and advanced endoscopic resection techniques. Further research is required to assess long-term outcomes in surveilled R1 cases, understand optimal endoscopic resection techniques and further develop local surveillance guidelines.

Enhancing Patient Care With the "David and Wendy" Approach: The Effectiveness of Ward Round Documentation Proformas.

Background Surgical ward round documentation, essential for high-quality patient care, is often completed poorly. The advent of electronic medical records offers an opportunity to introduce proformas, aiding junior staff in completing notes both timely and accurately. We aimed to assess whether the introduction of a proforma would improve the quality and speed of ward round documentation. Methods We completed a prospective cohort analysis of ward round documentation at a single institution. Analysis was conducted on the documentation of a single surgical team over a 10-week period, comprising five weeks of baseline data collection followed by five weeks with implementation of a proforma. This proforma was based on the "David & Wendy" acronym, encompassing diet, activity, vital signs, investigations/IV therapy, drains/lines, wound assessment, examination findings, nursing concerns, drugs/deep vein thrombosis (DVT) prophylaxis, and barriers to discharge. Results A total of 711 ward round notes were analyzed, 349 with proforma and 362 without. Statistically significant improvements were observed in the documentation of diet, activity, investigations/IV therapy, drains/lines, wound assessment, nursing concerns, drugs/DVT prophylaxis, and barriers to discharge (p < 0.05) with proforma use. No significant difference was noted in the documentation of vital signs or examination findings. The time taken to finalize ward round notes was significantly reduced with the proforma (M = 31.28 vs. 60.05 minutes, p < 0.001). Conclusion The introduction of the David & Wendy proforma significantly improved the speed and quality of documentation for key surgical ward round information during our study.

Acute copper deficiency myelopathy after single-anastomosis gastric bypass.

Bariatric surgery is a well-established treatment for morbid obesity, combining both restrictive and malabsorptive mechanisms to achieve weight loss. Macro and micronutrient deficiencies are some of the most common complications of these operations, which in rare occasions can be unexpected, severe, and difficult to manage. We present a case of severe copper deficiency related myelopathy in a patient post single anastomosis gastric bypass, requiring parenteral copper replacement and eventual reversal. She presented with ascending lower limb paraesthesia and weakness, with copper levels on admission of 4 µmol/l, and ceruloplasmin 94 mg/l. She continued to have progressive neuropathy and visual deterioration, despite IV and enteral replacement, and eventually underwent reversal of her bypass, with normalization in her copper levels and incomplete improvement in symptoms. Copper deficiency myelopathy is a rare and severe complication of bariatric surgery. Early identification is key, as neurological symptoms are often not reversible.

Granulins rescue inflammation, lysosome dysfunction, and neuropathology in a mouse model of progranulin deficiency.

Progranulin (PGRN) deficiency is linked to neurodegenerative diseases including frontotemporal dementia, Alzheimer's disease, Parkinson's disease, and neuronal ceroid lipofuscinosis. Proper PGRN levels are critical to maintain brain health and neuronal survival, however the function of PGRN is not well understood. PGRN is composed of 7.5 tandem repeat domains, called granulins, and is proteolytically processed into individual granulins inside the lysosome. The neuroprotective effects of full-length PGRN are well-documented, but the role of granulins is still unclear. Here we report, for the first time, that expression of single granulins is sufficient to rescue the full spectrum of disease pathology in mice with complete PGRN deficiency (Grn-/-). Specifically, rAAV delivery of either human granulin-2 or granulin-4 to Grn-/- mouse brain ameliorates lysosome dysfunction, lipid dysregulation, microgliosis, and lipofuscinosis similar to full-length PGRN. These findings support the idea that individual granulins are the functional units of PGRN, likely mediate neuroprotection within the lysosome, and highlight their importance for developing therapeutics to treat FTD-GRN and other neurodegenerative diseases.

Proximity-based labeling reveals DNA damage-induced phosphorylation of fused in sarcoma (FUS) causes distinct changes in the FUS protein interactome.

Accumulation of cytoplasmic inclusions containing fused in sarcoma (FUS), an RNA/DNA-binding protein, is a common hallmark of frontotemporal lobar degeneration and amyotrophic lateral sclerosis neuropathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 aired with mass spectrometry to investigate whether N-terminal phosphorylation alters the FUS protein-protein interaction network (interactome), and subsequently, FUS function. We report the first analysis comparing the interactomes of three FUS variants: homeostatic wildtype FUS (FUS WT), phosphomimetic FUS (FUS PM; a proxy for N-terminally phosphorylated FUS), and the toxic FUS proline 525 to leucine mutant (FUS P525L) that causes juvenile amyotrophic lateral sclerosis. We found that the phosphomimetic FUS interactome is uniquely enriched for a group of cytoplasmic proteins that mediate mRNA metabolism and translation, as well as nuclear proteins involved in the spliceosome and DNA repair functions. Furthermore, we identified and validated the RNA-induced silencing complex RNA helicase MOV10 as a novel interacting partner of FUS. Finally, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady-state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the interactome and function of FUS.

Network analysis of the progranulin-deficient mouse brain proteome reveals pathogenic mechanisms shared in human frontotemporal dementia caused by GRN mutations.

Heterozygous, loss-of-function mutations in the granulin gene (GRN) encoding progranulin (PGRN) are a common cause of frontotemporal dementia (FTD). Homozygous GRN mutations cause neuronal ceroid lipofuscinosis-11 (CLN11), a lysosome storage disease. PGRN is a secreted glycoprotein that can be proteolytically cleaved into seven bioactive 6 kDa granulins. However, it is unclear how deficiency of PGRN and granulins causes neurodegeneration. To gain insight into the mechanisms of FTD pathogenesis, we utilized Tandem Mass Tag isobaric labeling mass spectrometry to perform an unbiased quantitative proteomic analysis of whole-brain tissue from wild type (Grn+/+) and Grn knockout (Grn-/-) mice at 3- and 19-months of age. At 3-months lysosomal proteins (i.e. Gns, Scarb2, Hexb) are selectively increased indicating lysosomal dysfunction is an early consequence of PGRN deficiency. Additionally, proteins involved in lipid metabolism (Acly, Apoc3, Asah1, Gpld1, Ppt1, and Naaa) are decreased; suggesting lysosomal degradation of lipids may be impaired in the Grn-/- brain. Systems biology using weighted correlation network analysis (WGCNA) of the Grn-/- brain proteome identified 26 modules of highly co-expressed proteins. Three modules strongly correlated to Grn deficiency and were enriched with lysosomal proteins (Gpnmb, CtsD, CtsZ, and Tpp1) and inflammatory proteins (Lgals3, GFAP, CD44, S100a, and C1qa). We find that lysosomal dysregulation is exacerbated with age in the Grn-/- mouse brain leading to neuroinflammation, synaptic loss, and decreased markers of oligodendrocytes, myelin, and neurons. In particular, GPNMB and LGALS3 (galectin-3) were upregulated by microglia and elevated in FTD-GRN brain samples, indicating common pathogenic pathways are dysregulated in human FTD cases and Grn-/- mice. GPNMB levels were significantly increased in the cerebrospinal fluid of FTD-GRN patients, but not in MAPT or C9orf72 carriers, suggesting GPNMB could be a biomarker specific to FTD-GRN to monitor disease onset, progression, and drug response. Our findings support the idea that insufficiency of PGRN and granulins in humans causes neurodegeneration through lysosomal dysfunction, defects in autophagy, and neuroinflammation, which could be targeted to develop effective therapies.

Divergent FUS phosphorylation in primate and mouse cells following double-strand DNA damage.

Fused in sarcoma (FUS) is a RNA/DNA protein involved in multiple nuclear and cytoplasmic functions including transcription, splicing, mRNA trafficking, and stress granule formation. To accomplish these many functions, FUS must shuttle between cellular compartments in a highly regulated manner. When shuttling is disrupted, FUS abnormally accumulates into cytoplasmic inclusions that can be toxic. Disrupted shuttling of FUS into the nucleus is a hallmark of ~10% of frontotemporal lobar degeneration (FTLD) cases, the neuropathology that underlies frontotemporal dementia (FTD). Multiple pathways are known to disrupt nuclear/cytoplasmic shuttling of FUS. In earlier work, we discovered that double-strand DNA breaks (DSBs) trigger DNA-dependent protein kinase (DNA-PK) to phosphorylate FUS (p-FUS) at N-terminal residues leading to the cytoplasmic accumulation of FUS. Therefore, DNA damage may contribute to the development of FTLD pathology with FUS inclusions. In the present study, we examined how DSBs effect FUS phosphorylation in various primate and mouse cellular models. All cell lines derived from human and non-human primates exhibit N-terminal FUS phosphorylation following calicheamicin γ1 (CLM) induced DSBs. In contrast, we were unable to detect FUS phosphorylation in mouse-derived primary neurons or immortalized cell lines regardless of CLM treatment, duration, or concentration. Despite DNA damage induced by CLM treatment, we find that mouse cells do not phosphorylate FUS, likely due to reduced levels and activity of DNA-PK compared to human cells. Taken together, our work reveals that mouse-derived cellular models regulate FUS in an anomalous manner compared to primate cells. This raises the possibility that mouse models may not fully recapitulate the pathogenic cascades that lead to FTLD with FUS pathology.

Chimeric Peptide Species Contribute to Divergent Dipeptide Repeat Pathology in c9ALS/FTD and SCA36.

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.

Intracellular Proteolysis of Progranulin Generates Stable, Lysosomal Granulins that Are Haploinsufficient in Patients with Frontotemporal Dementia Caused by GRN Mutations.

Homozygous or heterozygous mutations in the GRN gene, encoding progranulin (PGRN), cause neuronal ceroid lipofuscinosis (NCL) or frontotemporal dementia (FTD), respectively. NCL and FTD are characterized by lysosome dysfunction and neurodegeneration, indicating PGRN is important for lysosome homeostasis in the brain. PGRN is trafficked to the lysosome where its functional role is unknown. PGRN can be cleaved into seven 6-kDa proteins called granulins (GRNs); however, little is known about how GRNs are produced or if levels of GRNs are altered in FTD-GRN mutation carriers. Here, we report the identification and characterization of antibodies that reliably detect several human GRNs by immunoblot and immunocytochemistry. Using these tools, we find that endogenous GRNs are present within multiple cell lines and are constitutively produced. Further, extracellular PGRN is endocytosed and rapidly processed into stable GRNs within lysosomes. Processing of PGRN into GRNs is conserved between humans and mice and is modulated by sortilin expression and mediated by cysteine proteases (i.e. cathpesin L). Induced lysosome dysfunction caused by alkalizing agents or increased expression of transmembrane protein 106B (TMEM106B) inhibit processing of PGRN into GRNs. Finally, we find that multiple GRNs are haploinsufficient in primary fibroblasts and cortical brain tissue from FTD-GRN patients. Taken together, our findings raise the interesting possibility that GRNs carry out critical lysosomal functions and that loss of GRNs should be explored as an initiating factor in lysosomal dysfunction and neurodegeneration caused by GRN mutations.

Disulfide bonds and disorder in granulin-3: An unusual handshake between structural stability and plasticity.

Granulins (GRNs) are a family of small (∼6 kDa) proteins generated by the proteolytic processing of their precursor, progranulin (PGRN), in many cell types. Both PGRN and GRNs are implicated in a plethora of biological functions, often in opposing roles to each other. Lately, GRNs have generated significant attention due to their implicated roles in neurodegenerative disorders. Despite their physiological and pathological significance, the structure-function relationships of GRNs are poorly defined. GRNs contain 12 conserved cysteines forming six intramolecular disulfide bonds, making them rather exceptional, even among a few proteins with high disulfide bond density. Solution NMR investigations in the past have revealed a unique structure containing putative interdigitated disulfide bonds for several GRNs, but GRN-3 was unsolvable due to its heterogeneity and disorder. In our previous report, we showed that abrogation of disulfide bonds in GRN-3 renders the protein completely disordered (Ghag et al., Prot Eng Des Sel 2016). In this study, we report the cellular expression and biophysical analysis of fully oxidized, native GRN-3. Our results indicate that both E. coli and human embryonic kidney (HEK) cells do not exclusively make GRN-3 with homogenous disulfide bonds, likely due to the high cysteine density within the protein. Biophysical analysis suggests that GRN-3 structure is dominated by irregular loops held together only by disulfide bonds, which induced remarkable thermal stability to the protein despite the lack of regular secondary structure. This unusual handshake between disulfide bonds and disorder within GRN-3 could suggest a unique adaptation of intrinsically disordered proteins towards structural stability.

Trehalose upregulates progranulin expression in human and mouse models of GRN haploinsufficiency: a novel therapeutic lead to treat frontotemporal dementia.

BACKGROUND: Progranulin (PGRN) is a secreted growth factor important for neuronal survival and may do so, in part, by regulating lysosome homeostasis. Mutations in the PGRN gene (GRN) are a common cause of frontotemporal lobar degeneration (FTLD) and lead to disease through PGRN haploinsufficiency. Additionally, complete loss of PGRN in humans leads to neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Importantly, Grn-/- mouse models recapitulate pathogenic lysosomal features of NCL. Further, GRN variants that decrease PGRN expression increase the risk of developing Alzheimer's disease (AD) and Parkinson's disease (PD). Together these findings demonstrate that insufficient PGRN predisposes neurons to degeneration. Therefore, compounds that increase PGRN levels are potential therapeutics for multiple neurodegenerative diseases. RESULTS: Here, we performed a cell-based screen of a library of known autophagy-lysosome modulators and identified multiple novel activators of a human GRN promoter reporter including several common mTOR inhibitors and an mTOR-independent activator of autophagy, trehalose. Secondary cellular screens identified trehalose, a natural disaccharide, as the most promising lead compound because it increased endogenous PGRN in all cell lines tested and has multiple reported neuroprotective properties. Trehalose dose-dependently increased GRN mRNA as well as intracellular and secreted PGRN in both mouse and human cell lines and this effect was independent of the transcription factor EB (TFEB). Moreover, trehalose rescued PGRN deficiency in human fibroblasts and neurons derived from induced pluripotent stem cells (iPSCs) generated from GRN mutation carriers. Finally, oral administration of trehalose to Grn haploinsufficient mice significantly increased PGRN expression in the brain. CONCLUSIONS: This work reports several novel autophagy-lysosome modulators that enhance PGRN expression and identifies trehalose as a promising therapeutic for raising PGRN levels to treat multiple neurodegenerative diseases.

FUS is phosphorylated by DNA-PK and accumulates in the cytoplasm after DNA damage.

Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) in neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). FUS is a member of the FET protein family that includes Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 2N (TAF15). FET proteins are predominantly localized to the nucleus, where they bind RNA and DNA to modulate transcription, mRNA splicing, and DNA repair. In ALS cases with FUS inclusions (ALS-FUS), mutations in the FUS gene cause disease, whereas FTLD cases with FUS inclusions (FTLD-FUS) do not harbor FUS mutations. Notably, in FTLD-FUS, all FET proteins accumulate with their nuclear import receptor Transportin 1 (TRN1), in contrast ALS-FUS inclusions are exclusively positive for FUS. In the present study, we show that induction of DNA damage replicates several pathologic hallmarks of FTLD-FUS in immortalized human cells and primary human neurons and astrocytes. Treatment with the antibiotic calicheamicin γ1, which causes DNA double-strand breaks, leads to the cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1. Moreover, cytoplasmic translocation of FUS is mediated by phosphorylation of its N terminus by the DNA-dependent protein kinase. Finally, we observed elevated levels of phospho-H2AX in FTLD-FUS brains, indicating that DNA damage occurs in patients. Together, our data reveal a novel regulatory mechanism for FUS localization in cells and suggest that DNA damage may contribute to the accumulation of FET proteins observed in human FTLD-FUS cases, but not in ALS-FUS.

Progranulin does not bind tumor necrosis factor (TNF) receptors and is not a direct regulator of TNF-dependent signaling or bioactivity in immune or neuronal cells.

Progranulin (PGRN) is a secreted glycoprotein expressed in neurons and glia that is implicated in neuronal survival on the basis that mutations in the GRN gene causing haploinsufficiency result in a familial form of frontotemporal dementia (FTD). Recently, a direct interaction between PGRN and tumor necrosis factor receptors (TNFR I/II) was reported and proposed to be a mechanism by which PGRN exerts anti-inflammatory activity, raising the possibility that aberrant PGRN-TNFR interactions underlie the molecular basis for neuroinflammation in frontotemporal lobar degeneration pathogenesis. Here, we report that we find no evidence for a direct physical or functional interaction between PGRN and TNFRs. Using coimmunoprecipitation and surface plasmon resonance (SPR) we replicated the interaction between PGRN and sortilin and that between TNF and TNFRI/II, but not the interaction between PGRN and TNFRs. Recombinant PGRN or transfection of a cDNA encoding PGRN did not antagonize TNF-dependent NFκB, Akt, and Erk1/2 pathway activation; inflammatory gene expression; or secretion of inflammatory factors in BV2 microglia and bone marrow-derived macrophages (BMDMs). Moreover, PGRN did not antagonize TNF-induced cytotoxicity on dopaminergic neuroblastoma cells. Last, co-addition or pre-incubation with various N- or C-terminal-tagged recombinant PGRNs did not alter lipopolysaccharide-induced inflammatory gene expression or cytokine secretion in any cell type examined, including BMDMs from Grn+/- or Grn-/- mice. Therefore, the neuroinflammatory phenotype associated with PGRN deficiency in the CNS is not a direct consequence of the loss of TNF antagonism by PGRN, but may be a secondary response by glia to disrupted interactions between PGRN and Sortilin and/or other binding partners yet to be identified.

Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis.

BACKGROUND: Mutations in the gene encoding the RNA-binding protein fused in sarcoma (FUS) can cause familial and sporadic amyotrophic lateral sclerosis (ALS) and rarely frontotemproal dementia (FTD). FUS accumulates in neuronal cytoplasmic inclusions (NCIs) in ALS patients with FUS mutations. FUS is also a major pathologic marker for a group of less common forms of frontotemporal lobar degeneration (FTLD), which includes atypical FTLD with ubiquitinated inclusions (aFTLD-U), neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD). These diseases are now called FUS proteinopathies, because they share this disease marker. It is unknown how FUS mutations cause disease and the role of FUS in FTD-FUS cases, which do not have FUS mutations. In this paper we report the development of somatic brain transgenic (SBT) mice using recombinant adeno-associated virus (rAAV) to investigate how FUS mutations lead to neurodegeneration. RESULTS: We compared SBT mice expressing wild-type human FUS (FUSWT), and two ALS-linked mutations: FUSR521C and FUSΔ14, which lacks the nuclear localization signal. Both FUS mutants accumulated in the cytoplasm relative to FUSWT. The degree of this shift correlated with the severity of the FUS mutation as reflected by disease onset in humans. Mice expressing the most aggressive mutation, FUSΔ14, recapitulated many aspects of FUS proteinopathies, including insoluble FUS, basophilic and eosiniphilic NCIs, and other pathologic markers, including ubiquitin, p62/SQSTM1, α-internexin, and the poly-adenylate(A)-binding protein 1 (PABP-1). However, TDP-43 did not localize to inclusions. CONCLUSIONS: Our data supports the hypothesis that ALS or FTD-linked FUS mutations cause neurodegeneration by increasing cyotplasmic FUS. Accumulation of FUS in the cytoplasm may retain RNA targets and recruit additional RNA-binding proteins, such as PABP-1, into stress-granule like aggregates that coalesce into permanent inclusions that could negatively affect RNA metabolism. Identification of mutations in other genes that cause ALS/FTD, such as C9ORF72, sentaxin, and angiogenin, lends support to the idea that defective RNA metabolism is a critical pathogenic pathway. The SBT FUS mice described here will provide a valuable platform for dissecting the pathogenic mechanism of FUS mutations, define the relationship between FTD and ALS-FUS, and help identify therapeutic targets that are desperately needed for these devastating neurodegenerative disorders.

Parkinson's disease is not associated with gastrointestinal myenteric ganglion neuron loss.

Gastrointestinal dysfunction is a prominent non-motor feature of Parkinson's disease (PD) that contributes directly to the morbidity of patients, complicates management of motor symptoms, and may herald incipient PD in patients without motor disability. Although PD has traditionally been considered a disease of dopaminergic neurons in the substantia nigra, analyses of gastrointestinal samples from PD patients have consistently revealed pathology in the enteric nervous system. The relationship of PD pathology to GI dysmotility is poorly understood, and this lack of understanding has led to limited success in developing treatments for PD-related GI symptoms. We have quantitatively compared myenteric neuron density and relative abundance of NO, VIP, and catecholamine neurons between patients with PD and control individuals along the length of the GI tract. In addition, we have examined the frequency of GI α-synuclein neuritic pathology and its co-localization with the same neuronal markers. We have included a comparison with a small population of patients with incidental Lewy bodies found at autopsy. These data indicate that there is no neuronal loss in the myenteric plexus in PD. Lewy body pathology parallels parasympathetic autonomic input from the dorsal motor nucleus of the vagus, not the distribution of extrinsic sympathetic input or intrinsic enteric neurons, and is only rarely co-localized with tyrosine hydroxylase. These data provide a critical background to which further analyses of the effect of PD on the GI tract may be compared and suggest that neuropathology in myenteric neurons is unlikely to be a causative factor in PD-related GI dysmotility.

Alpha-synuclein transgenic mice display age-related slowing of gastrointestinal motility associated with transgene expression in the vagal system.

Gastrointestinal (GI) dysfunction is the one of the most common non-motor symptoms of Parkinson's disease (PD) and occurs in nearly every patient afflicted with this common neurodegenerative disorder. While parkinsonian motor symptoms are caused by degeneration of dopamine neurons in the midbrain substantia nigra, the neurological localization of non-motor symptoms in PD is not known. In this study, we examined a transgenic mouse model of PD in which mutant (A53T) human α-synuclein was expressed under control of the prion promoter (AS mice). We found that gastrointestinal expression of human α-synuclein in this transgenic line was limited to efferent fibers projecting from the dorsal motor nucleus of the vagus nerve (DMV) to the enteric nervous system (ENS). Older transgenic mice had a lower density of human α-synuclein expression in the GI tract, suggesting an age-related disruption of efferent vagal fibers in this model. At the same time, mice developed age-related declines in stool frequency and gastric emptying consistent with those seen in human PD. These behavioral and neuropathological patterns parallel those seen in PD patients and suggest the DMV as a target for further investigation into causes for GI neuropathology and symptomatology in parkinsonism.

Neurochemical phenotypes of myenteric neurons in the rhesus monkey.

Understanding the neurochemical composition of the enteric nervous system (ENS) is critical for elucidating neurological function in the gastrointestinal (GI) tract in health and disease. Despite their status as the closest models of human neurological systems, relatively little is known about enteric neurochemistry in nonhuman primates. We describe neurochemical coding of the enteric nervous system, specifically the myenteric plexus, of the rhesus monkey (Macaca mulatta) by immunohistochemistry and directly compare it to human tissues. There are considerable differences in the myenteric plexus along different segments of the monkey GI tract. While acetylcholine neurons make up the majority of myenteric neurons in the stomach (70%), they are a minority in the rectum (47%). Conversely, only 22% of gastric myenteric neurons express nitric oxide synthase (NOS) compared to 52% in the rectum. Vasoactive intestinal peptide (VIP) is more prominent in the stomach (37%) versus the rest of the GI tract (≈10%), and catecholamine neurons are rare (≈1%). There is significant coexpression of NOS and VIP in myenteric neurons that is more prominent in the proximal GI tract. Taken as a whole, these data provide insight into the neurochemical anatomy underlying GI motility. While overall similarity to other mammalian species is clear, there are some notable differences between the ENS of rhesus monkeys, humans, and other species that will be important to take into account when evaluating models of human diseases in animals.

A novel transferrin/TfR2-mediated mitochondrial iron transport system is disrupted in Parkinson's disease.

More than 80 years after iron accumulation was initially described in the substantia nigra (SN) of Parkinson's disease (PD) patients, the mechanisms responsible for this phenomenon are still unknown. Similarly, how iron is delivered to its major recipients in the cell - mitochondria and the respiratory complexes - has yet to be elucidated. Here, we report a novel transferrin/transferrin receptor 2 (Tf/TfR2)-mediated iron transport pathway in mitochondria of SN dopamine neurons. We found that TfR2 has a previously uncharacterized mitochondrial targeting sequence that is sufficient to import the protein into these organelles. Importantly, the Tf/TfR2 pathway can deliver Tf bound iron to mitochondria and to the respiratory complex I as well. The pathway is redox-sensitive and oxidation of Tf thiols to disulfides induces release from Tf of highly reactive ferrous iron, which contributes to free radical production. In the rotenone model of PD, Tf accumulates in dopamine neurons, with much of it accumulating in the mitochondria. This is associated with iron deposition in SN, similar to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention.