With the increased use of small self-complementary adeno-associated viral (AAV) vectors, the design of compact promoters becomes critical for packaging and expressing larger transgenes under ubiquitous or cell-specific control. In a comparative study of commonly used 800-bp cytomegalovirus (CMV) and chicken β-actin (CBA) promoters, we report significant differences in the patterns of cell-specific gene expression in the central and peripheral nervous systems. The CMV promoter provides high initial neural expression that diminishes over time. The CBA promoter displayed mostly ubiquitous and high neural expression, but substantially lower expression in motor neurons (MNs). We report the creation of a novel hybrid form of the CBA promoter (CBh) that provides robust long-term expression in all cells observed with CMV or CBA, including MNs. To develop a short neuronal promoter to package larger transgenes into AAV vectors, we also found that a 229-bp fragment of the mouse methyl-CpG-binding protein-2 (MeCP2) promoter was able to drive neuron-specific expression within the CNS. Thus the 800-bp CBh promoter provides strong, long-term, and ubiquitous CNS expression whereas the MeCP2 promoter allows an extra 570-bp packaging capacity, with low and mostly neuronal expression within the CNS, similar to the MeCP2 transcription factor.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTD cases into at least four different subtypes, which correlate with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for extremely pure biochemical isolation of pathological TDP-43. Combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43, which become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from disease brain forms large and stable assemblies of distinct densities and morphologies that correlate with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies displayed differential neurotoxicity and seeding that correlated with disease duration of FTLD patients. Our data indicate that disease heterogeneity may originate from alternate pathological TDP-43 conformations, reminiscent of prion strains. 5 developed SarkoSpin, a novel and simple extraction method for physical separation of pathological TDP-43 from more than 99% of total protein mass including the extreme bulk of physiological, monomeric and oligomeric 11 TDP-43. Using SarkoSpin on brain cortical samples from over 80 patients, we found that TDP-43 forms large and buoyant assemblies of distinct densities, polyubiquitination levels and morphologies that correlate with specific neuropathological classifications. Importantly, coupling SarkoSpin with mass spectrometry, we illustrate that a specific subset of proteins, beyond TDP-43, become insoluble in each disease subtype. These proteins are rarely co-aggregated with pTDP-43 and most likely represent a downstream effect of TDP-43 pathology. One of these proteins depicts a distinct astrocytic reaction discriminating FTLD-TDP-A from FTLD-TDP-C patients, illustrating divergent pathogenic mechanisms within these two disease subtypes. Most importantly, we show evidence that SarkoSpin extracted pTDP-43 assemblies exhibit cytotoxicity and protein seeding ability. Remarkably, pathological aggregates extracted from FTLD-TDP-A were significantly more cyto-and neurotoxic than those extracted from FTLD-TDP-C, thereby correlating with the significant difference in disease duration between these two subtypes. Collectively, our data demonstrate that ALS and FTLD heterogeneity is consistently reflected in the biochemical, neurotoxic and seeding properties of TDP-43 and the associated insoluble proteome. We propose that alternative TDP-43 pathological conformations may underlie the diversity of TDP-43 proteinopathies, reminiscent of prion strains 33,34. Results Summary of patient cohort and characterization of FTLD-TDP-A and FTLD-TDP-C cases Brain cortical samples from over 80 patients, including control patients with no apparent CNS pathology or with non-TDP...

Excess neural activity in the CA3 region of the hippocampus has been linked to memory impairment in aged rats. We tested whether interventions aimed at reducing this excess activity would improve memory performance. Aged (24–28 mo-old) male Long-Evans rats were characterized in a spatial memory task known to depend on the functional integrity of the hippocampus, such that aged rats with identified memory impairment were used in a series of experiments. Over-expression of the inhibitory neuropeptide Y 13–36 in the CA3 via adeno-associated viral transduction was found to improve hippocampal-dependent long-term memory in aged rats that had been characterized with impairment. Subsequent experiments with two commonly used antiepileptic agents, sodium valproate and levetiracetam, similarly produced dose-dependent memory improvement in such aged rats. Improved spatial memory with low doses of these agents was observed in both appetitve and aversive spatial tasks. The benefits of these different modalities of treatment are consistent with the concept that excess activity in the CA3 region of the hippocampus is a dysfunctional condition that may play a key role underlying age-related impairment in hippocampal-dependent memory processes. Because increased hippocampal activation occurs in age-related memory impairment in humans as observed in functional neuroimaging, the current findings also suggest that low doses of certain antiepileptic drugs in cognitively impaired elderly humans may have therapeutic potential and point to novel targets for this indication.

Clinical diagnosis of multiple system atrophy is challenging and many patients with Lewy body disease (i.e. Parkinson’s disease or dementia with Lewy bodies) or progressive supranuclear palsy are misdiagnosed as having multiple system atrophy in life. The clinical records of 203 patients with a clinical diagnosis of multiple system atrophy were reviewed to identify diagnostic pitfalls. We also examined 12 features supporting a diagnosis of multiple system atrophy (red flag features: orofacial dystonia, disproportionate antecollis, camptocormia and/or Pisa syndrome, contractures of hands or feet, inspiratory sighs, severe dysphonia, severe dysarthria, snoring, cold hands and feet, pathological laughter and crying, jerky myoclonic postural/action tremor and polyminimyoclonus) and seven disability milestones (frequent falls, use of urinary catheters, wheelchair dependent, unintelligible speech, cognitive impairment, severe dysphagia, residential care). Of 203 cases, 160 (78.8%) were correctly diagnosed in life and had pathologically confirmed multiple system atrophy. The remaining 21.2% (43/203) had alternative pathological diagnoses including Lewy body disease (12.8%; n = 26), progressive supranuclear palsy (6.4%; n = 13), cerebrovascular diseases (1%; n = 2), amyotrophic lateral sclerosis (0.5%; n = 1) and cerebellar degeneration (0.5%; n = 1). More patients with multiple system atrophy developed ataxia, stridor, dysphagia and falls than patients with Lewy body disease; resting tremor, pill-rolling tremor and hallucinations were more frequent in Lewy body disease. Although patients with multiple system atrophy and progressive supranuclear palsy shared several symptoms and signs, ataxia and stridor were more common in multiple system atrophy. Multiple logistic regression analysis revealed increased likelihood of multiple system atrophy versus Lewy body disease and progressive supranuclear palsy if a patient developed orthostatic hypotension or urinary incontinence with the requirement for urinary catheters [multiple system atrophy versus Lewy body disease: odds ratio (OR): 2.0, 95% confidence interval (CI): 1.1–3.7, P = 0.021; multiple system atrophy versus progressive supranuclear palsy: OR: 11.2, 95% CI: 3.2–39.2, P < 0.01]. Furthermore, autonomic dysfunction within the first 3 years from onset can differentiate multiple system atrophy from progressive supranuclear palsy (multiple system atrophy versus progressive supranuclear palsy: OR: 3.4, 95% CI: 1.2–9.7, P = 0.023). Multiple system atrophy patients with predominant parkinsonian signs had a higher number of red flag features than patients with Lewy body disease (OR: 8.8, 95% CI: 3.2–24.2, P < 0.01) and progressive supranuclear palsy (OR: 4.8, 95% CI: 1.7–13.6, P < 0.01). The number of red flag features in multiple system atrophy with predominant cerebellar signs was also higher than in Lewy body disease (OR: 7.0, 95% CI: 2.5–19.5, P < 0.01) and progressive supranuclear palsy (OR: 3.1, 95% CI: 1.1–8.9, P = 0.032). Patients with multiple system atrophy had shorter latency to reach use of urinary catheter and longer latency to residential care than progressive supranuclear palsy patients, whereas patients with Lewy body disease took longer to reach multiple milestones than patients with multiple system atrophy. The present study has highlighted features which should improve the ante-mortem diagnostic accuracy of multiple system atrophy.

The neurodegenerative disorder Alzheimer's disease is characterised by the formation of β-amyloid plaques and neurofibrillary tangles in the brain parenchyma, which cause synapse and neuronal loss. This leads to clinical symptoms, such as progressive memory deficits. Clinically, these pathological changes can be detected in the cerebrospinal fluid and with brain imaging, although reliable blood tests for plaque and tangle pathologies remain to be developed. Plaques and tangles often co-exist with other brain pathologies, including aggregates of transactive response DNA-binding protein 43 and Lewy bodies, but the extent to which these contribute to the severity of Alzheimer's disease is currently unknown. In this ‘At a glance’ article and poster, we summarise the molecular biomarkers that are being developed to detect Alzheimer's disease and its related pathologies. We also highlight the biomarkers that are currently in clinical use and include a critical appraisal of the challenges associated with applying these biomarkers for diagnostic and prognostic purposes of Alzheimer's disease and related neurodegenerative disorders, also in their prodromal clinical phases.