AAV-based delivery of RNAi targeting Ataxin-2 improves survival, strength, and pathology in mouse models of rapidly and slowly progressive sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron death due to nuclear loss and cytoplasmic aggregation of the splice factor TDP-43. Pathologic TDP-43 associates with stress granules (SGs) and downregulating the SG-associated protein Ataxin-2 (Atxn2) using antisense oligonucleotides (ASO) prolongs survival in the TAR4/4 sporadic ALS mouse model, a strategy now in clinical trials. Here, we used AAV-mediated RNAi delivery to achieve lasting and targeted Atxn2 knockdown after a single injection. To achieve this, a novel AAV with improved transduction potency of our target cells was used to deliver Atxn2 -targeting miRNAs. Mouse dosing studies demonstrated 55% Atxn2 knockdown in frontal cortex and 25% knockdown throughout brainstem and spinal cord after intracerebroventricular injection at a dose 40x lower than used in other recent studies. In TAR4/4 mice, miAtxn2 treatment increased mean and median survival by 54% and 45% respectively (p<0.0003). Mice showed robust improvement across strength-related measures ranging from 24-75%. Interestingly, treated mice showed increased vertical activity above wildtype, suggesting unmasking of an FTD phenotype with improved strength. Histologically, lower motor neuron survival improved with a concomitant reduction in CNS inflammatory markers. Additionally, phosphorylated TDP-43 was reduced to wildtype levels. Bulk RNA sequencing revealed correction of 153 genes in the markedly dysregulated transcriptome of mutant mice, several of which are described in the human ALS literature. In slow progressing hemizygous mice, treatment rescued weight loss and improved gait at late time points. Cumulatively the data support the utility of AAV-mediated RNAi against Atxn2 as a robust and translatable treatment strategy for sporadic ALS.

Interactive euthanasia training program for the dairy industry: Implementation and perceived respondent knowledge change.

This study described the development of an interactive euthanasia training program and its potential to improve dairy workers' perceived euthanasia decision-making skills and awareness of timely euthanasia by using a survey instrument before and after the program. Training material encompassed euthanasia information over 2 production stages (calves and cows or heifers) and material was delivered on-farm in a case-scenario format (14 cases). During a 3-mo period, 30 different dairy farms were visited and 81 participants were enrolled in this study. Each participant was required to complete a survey pretraining, to complete the case studies from the production stage in which their job responsibility was more closely aligned with (estimated completion time of 1 h), and to complete a survey post-training. Surveys contained 8 statements regarding participants' perceived knowledge of euthanasia practices. The questions were answered on a 5-point scale: (1) strongly disagree, (2) disagree, (3) neither agree nor disagree, (4) agree, or (5) strongly agree. Multivariable mixed-effects logistic regression models were created for each question to investigate the effect of age, sex, dairy experience, farm size, role at the farm, race, previous experience with euthanasia, veterinarian degree, and production stage in the score change, defined by the presence or absence of an increase in the 5-point scale score. Upon completion of the training, respondents were more confident in identifying compromised animals (score change = 0.35), determining when an animal should be euthanized (score change = 0.64), and understanding the importance of timely euthanasia (score change = 0.26). Age and euthanasia experience were significantly associated with the respondents' perceived knowledge; suggesting that younger, less-experienced caretakers on-farm should be prioritized to receive training. The proposed interactive case-based euthanasia training program has proven to be valuable to dairy participants and veterinarians as it provides a means to improve dairy welfare.

Dairy workers' attitudes toward dairy cattle euthanasia.

Implementing timely and humane euthanasia in dairy farms remains a critical concern. One of the possible barriers for the implementation of timely euthanasia on-farm is dairy workers' attitudes toward the act. The objectives of this study were to investigate dairy workers' attitudes toward dairy cattle euthanasia and their association to individuals' demographic characteristics. A total of 81 workers from 30 dairy farms (ranging in size from less than 500 to more than 3,000 cows) participated in the survey and most participants were caretakers (n = 45; 55.6%) or farm managers (n = 16; 19.8%), with an average work experience of 14.8 years. Dairy workers' attitudes toward dairy cattle (empathy affect, empathy attribution, and negative attitudes about cattle), working environment (relying on others, perceived time constraints) and euthanasia decision-making (feeling comfortable with euthanasia, feeling confident, seeking knowledge, using different sources to obtain advice, having negative attitudes about euthanasia, having insufficient knowledge, having trouble deciding when to euthanize and avoiding if possible) were identified and used for cluster analyses. Cluster analyses identified 3 distinct clusters: (1) confident but uncomfortable with euthanasia (n = 40); (2) confident and comfortable with euthanasia (n = 32); and (3) unconfident, lacking knowledge and detached from cattle (n = 9). Dairy workers' demographic characteristics (age, sex, race and ethnicity, dairy experience, role on-farm, farm size, and previous euthanasia experience) were used as predictors for the risk factor analyses. The risk analysis demonstrated that there were no predictors for cluster 1, but White workers (P = 0.04) and caretakers that had previous euthanasia experience tended to be more likely to be members of cluster 2 (P = 0.07) whereas respondents that worked in farms with 501-1,000 cows were more likely to be grouped in cluster 3. This study provides vital information about variability in dairy workers' attitudes toward dairy euthanasia as well as its association with race and ethnicity, farm size, and previous euthanasia experience. This information can be used to implement appropriate training and euthanasia protocols to increase both human and dairy cattle welfare on-farm.

Combined overexpression of ATXN1L and mutant ATXN1 knockdown by AAV rescue motor phenotypes and gene signatures in SCA1 mice.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease caused by a (CAG) repeat expansion in the coding sequence of ATXN1. The primary mechanism of disease in SCA1 is toxic gain of function by polyglutamine-expanded mutant ATXN1 and is compounded by partial loss of wild-type function. Addressing both disease mechanisms, we have shown that virally expressed RNA interference targeting ATXN1 can both prevent and reverse disease phenotypes in SCA1 mice, and that overexpression of the ATXN1 homolog, ataxin 1-like (ATXN1L), improves disease readouts when delivered pre-symptomatically. Here, we combined these therapeutic approaches into two, dual component recombinant adeno-associated virus (rAAV) vectors and tested their ability to reverse disease in symptomatic SCA1 mice using behavior, pathological, and next-generation sequencing assays. Mice treated with vectors expressing human ATXN1L (hATXN1L) alone showed motor improvements and changes in gene expression that reflected increases in pro-development pathways. When hATN1L was combined with miS1, a previously validated microRNA targeting h ATXN1, there was added normalization of disease allele-induced changes in gene expression along with motor improvements. Our data show the additive nature of this two-component approach for a more effective SCA1 therapy.

Persistent repression of tau in the brain using engineered zinc finger protein transcription factors.

Neuronal tau reduction confers resilience against ß-amyloid and tau-related neurotoxicity in vitro and in vivo. Here, we introduce a novel translational approach to lower expression of the tau gene MAPT at the transcriptional level using gene-silencing zinc finger protein transcription factors (ZFP-TFs). Following a single administration of adeno-associated virus (AAV), either locally into the hippocampus or intravenously to enable whole-brain transduction, we selectively reduced tau messenger RNA and protein by 50 to 80% out to 11 months, the longest time point studied. Sustained tau lowering was achieved without detectable off-target effects, overt histopathological changes, or molecular alterations. Tau reduction with AAV ZFP-TFs was able to rescue neuronal damage around amyloid plaques in a mouse model of Alzheimer's disease (APP/PS1 line). The highly specific, durable, and controlled knockdown of endogenous tau makes AAV-delivered ZFP-TFs a promising approach for the treatment of tau-related human brain diseases.

Tau reduction in aged mice does not impact Microangiopathy.

Microangiopathy, including proliferation of small diameter capillaries, increasing vessel tortuosity, and increased capillary blockage by leukocytes, was previously observed in the aged rTg4510 mouse model. Similar gene expression changes related to angiogenesis were observed in both rTg4510 and Alzheimer's disease (AD). It is uncertain if tau is directly responsible for these vascular changes by interacting directly with microvessels, and/or if it contributes indirectly via neurodegeneration and concurrent neuronal loss and inflammation. To better understand the nature of tau-related microangiopathy in human AD and in tau mice, we isolated capillaries and observed that bioactive soluble tau protein could be readily detected in association with vasculature. To examine whether this soluble tau is directly responsible for the microangiopathic changes, we made use of the tetracycline-repressible gene expression cassette in the rTg4510 mouse model and measured vascular pathology following tau reduction. These data suggest that reduction of tau is insufficient to alter established microvascular complications including morphological alterations, enhanced expression of inflammatory genes involved in leukocyte adherence, and blood brain barrier compromise. These data imply that 1) soluble bioactive tau surprisingly accumulates at the blood brain barrier in human brain and in mouse models, and 2) the morphological and molecular phenotype of microvascular disturbance does not resolve with reduction of whole brain soluble tau. Additional consideration of vascular-directed therapies and strategies that target tau in the vascular space may be required to restore normal function in neurodegenerative disease.

Experimental evidence for the age dependence of tau protein spread in the brain.

The incidence of Alzheimer's disease (AD), which is characterized by progressive cognitive decline that correlates with the spread of tau protein aggregation in the cortical mantle, is strongly age-related. It could be that age predisposes the brain for tau misfolding and supports the propagation of tau pathology. We tested this hypothesis using an experimental setup that allowed for exploration of age-related factors of tau spread and regional vulnerability. We virally expressed human tau locally in entorhinal cortex (EC) neurons of young or old mice and monitored the cell-to-cell tau protein spread by immunolabeling. Old animals showed more tau spreading in the hippocampus and adjacent cortical areas and accumulated more misfolded tau in EC neurons. No misfolding, at any age, was observed in the striatum, a brain region mostly unaffected by tangles. Age and brain region dependent tau spreading and misfolding likely contribute to the profound age-related risk for sporadic AD.

Partial reduction of microglia does not affect tau pathology in aged mice.

BACKGROUND: Activation of inflammation pathways in the brain occurs in Alzheimer's disease and may contribute to the accumulation and spread of pathological proteins including tau. The goal of this study was to identify how changes in microglia, a key inflammatory cell type, may contribute to tau protein accumulation and pathology-associated changes in immune and non-immune cell processes such as neuronal degeneration, astrocyte physiology, cytokine expression, and blood vessel morphology. METHODS: We used PLX3397 (290 mg/kg), a colony-stimulating factor receptor 1 (CSF1R) inhibitor, to reduce the number of microglia in the brains of a tau-overexpressing mouse model. Mice were fed PLX3397 in chow or a control diet for 3 months beginning at 12 months of age and then were subsequently analyzed for changes in blood vessel morphology by in vivo two-photon microscopy and tissues were collected for biochemistry and histology. RESULTS: PLX3397 reduced microglial numbers by 30% regardless of genotype compared to control diet-treated mice. No change in tau burden, cortical atrophy, blood vessels, or astrocyte activation was detected. All Tg4510 mice were observed to have an increased in "disease-associated" microglial gene expression, but PLX3397 treatment did not reduce expression of these genes. Surprisingly, PLX3397 treatment resulted in upregulation of CD68 and Tgf1ß. CONCLUSIONS: Manipulating microglial activity may not be an effective strategy to combat tau pathological lesions. Higher doses of PLX3397 may be required or earlier intervention in the disease course. Overall, this indicates a need for a better understanding of specific microglial changes and their relation to the disease process.

Tau induces blood vessel abnormalities and angiogenesis-related gene expression in P301L transgenic mice and human Alzheimer's disease.

Mixed pathology, with both Alzheimer's disease and vascular abnormalities, is the most common cause of clinical dementia in the elderly. While usually thought to be concurrent diseases, the fact that changes in cerebral blood flow are a prominent early and persistent alteration in Alzheimer's disease raises the possibility that vascular alterations and Alzheimer pathology are more directly linked. Here, we report that aged tau-overexpressing mice develop changes to blood vessels including abnormal, spiraling morphologies; reduced blood vessel diameters; and increased overall blood vessel density in cortex. Blood flow in these vessels was altered, with periods of obstructed flow rarely observed in normal capillaries. These changes were accompanied by cortical atrophy as well as increased expression of angiogenesis-related genes such as Vegfa, Serpine1, and Plau in CD31-positive endothelial cells. Interestingly, mice overexpressing nonmutant forms of tau in the absence of frank neurodegeneration also demonstrated similar changes. Furthermore, many of the genes we observe in mice are also altered in human RNA datasets from Alzheimer patients, particularly in brain regions classically associated with tau pathology such as the temporal lobe and limbic system regions. Together these data indicate that tau pathological changes in neurons can impact brain endothelial cell biology, altering the integrity of the brain's microvasculature.

Redesigning channel-forming peptides: amino acid substitutions that enhance rates of supramolecular self-assembly and raise ion transport activity.

Three series of 22-residue peptides derived from the transmembrane M2 segment of the glycine receptor alpha1-subunit (M2GlyR) have been designed, synthesized, and tested to determine the plasticity of a channel-forming sequence and to define whether channel pores with enhanced conductive properties could be created. Sixteen sequences were examined for aqueous solubility, solution-association tendency, secondary structure, and half-maximal concentration for supramolecular assembly, channel activity, and ion transport properties across epithelial monolayers. All peptides interact strongly with membranes: associating with, inserting across, and assembling to form homooligomeric bundles when in micromolar concentrations. Single and double amino acid replacements involving arginine and/or aromatic amino acids within the final five C-terminal residues of the peptide cause dramatic effects on the concentration dependence, yielding a range of K1/2 values from 36 +/- 5 to 390 +/- 220 microM for transport activity. New water/lipid interfacial boundaries were established for the transmembrane segment using charged or aromatic amino acids, thus limiting the peptides' ability to move perpendicularly to the plane of the bilayer. Formation of discrete water/lipid interfacial boundaries appears to be necessary for efficient supramolecular assembly and high anion transport activity. A peptide sequence is identified that may show efficacy in channel replacement therapy for channelopathies such as cystic fibrosis.

Activity and structural comparisons of solution associating and monomeric channel-forming peptides derived from the glycine receptor m2 segment.

A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor alpha(1) subunit (M2GlyR), including the 22-residue sequence NK(4)-M2GlyR p22 wild type (WT) (KKKKPARVGLGITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK(4)-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide adopts a bent conformation forming a hydrophobic pocket by hydrophobic interactions.