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Viewed as an imbalance between production and clearance of toxic Aβ peptides, Alzheimer’s disease is a candidate for therapies to augment brain waste removal. Prior work has shown that Aβ accumulates in meninges with aging as a byproduct of normal brain activity, in parallel with build-up of Aβ oligomers in neurons, blood vessels, and interstitial fluid. Using the TgF344-AD rat model of Alzheimer’s disease, we now report that dural lymphatic vessels specifically accumulate neurotoxic pyroglutamate amyloid beta (pE3-Aβ) with aging. Notably, accelerated amyloidosis is observed in meninges after ligation of cervical lymphatics, together with significantly increased pE3-Aβ and Aβ42 deposition in upstream brain regions implicated in Alzheimer’s disease. Blockade of lymphatic clearance is not sufficiently compensated by other efflux pathways, suggesting a necessary role of Aβ clearance at the level of lymphatics. We further report that dural lymphatic cells actively clear Aβ via energy-dependent mechanisms, and lymphatic Aβ transport is significantly impaired both in normal aging and in Alzheimer’s disease. Dural lymphatic cells isolated from the TgF344-AD rat show ultrastructural abnormalities in mitochondria and abnormal cytoplasmic inclusions, with a distinct transcriptional profile implicating failure of energy-dependent transport. Finally, using human meninges treated with FocusDeep tissue clearing, we demonstrate using whole mount panoramic imaging that dural lymphatic vessels comprise a structurally diverse intracranial vascular network that accumulates pE3-Aβ with aging, similar to the rat model. We conclude that intracranial meningeal and extracranial cervical lymphatic vessels are targets for Alzheimer’s disease therapies focused on improving amyloid clearance.One Sentence SummaryLymphatic vessels remove Aβ from the brain via energy-dependent active transport mechanisms, and blockage of extracranial lymphatic drainage is sufficient to cause significant acceleration of intracranial Alzheimer’s Aβ pathology in both meninges and brain.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects 1% of the population above the age of 60. The cause of the disease remains unknown. The histopathological hallmarks of the disease are intracytoplasmic Lewy bodies and dopaminergic striatal insufficiency secondary to a loss of dopaminergic neurons in the substantia nigra pars compacta (SN). Pharmacological treatment options for PD are often limited by the ability of prodopaminergic drugs to function within the nigrostriatal system, without activating other dopaminergic, but non-nigrostriatal regions, of the CNS. Even if this obstacle is overcome, considerations regarding the chronic availability of the drug can limit drug utility. Gene delivery systems are ideal for delivering therapeutic molecules to site specific regions of the CNS. Via gene therapy, a piece or pieces of DNA placed into a carrying vector encoding for a substance of interest is introduced into cells. While there are many ways to apply this technology, this review will focus on in vivo gene therapy as it applies to Parkinson's disease. Using stereotaxic surgery, vectors can be introduced into specific target areas in the brain and deliver genes encoding for therapeutic molecules. By delivering genes using gene therapy approaches, a therapeutic molecule can be delivered chronically in a site-specific fashion, diminishing unwanted side effects and repeated interventions to obtain useful levels of the drug. Throughout this review, we discuss the potential for gene delivery aimed at enhancing dopamine production or providing neuroprotection for nigrostriatal neurons to serve as a therapeutic strategy for PD

Lentiviral delivery of glial cell line-derived neurotrophic factor (lenti-GDNF) was tested for its trophic effects upon degenerating nigrostriatal neurons in nonhuman primate models of Parkinson's disease (PD). We injected lenti-GDNF into the striatum and substantia nigra of nonlesioned aged rhesus monkeys or young adult rhesus monkeys treated 1 week prior with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Extensive GDNF expression with anterograde and retrograde transport was seen in all animals. In aged monkeys, lenti-GDNF augmented dopaminergic function. In MPTP-treated monkeys, lenti-GDNF reversed functional deficits and completely prevented nigrostriatal degeneration. Additionally, lenti-GDNF injections to intact rhesus monkeys revealed long-term gene expression (8 months). In MPTP-treated monkeys, lenti-GDNF treatment reversed motor deficits in a hand-reach task. These data indicate that GDNF delivery using a lentiviral vector system can prevent nigrostriatal degeneration and induce regeneration in primate models of PD and might be a viable therapeutic strategy for PD patients.

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-minute and 1-hour postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129., Competing Interests: Declarations Competing interests The authors have no competing interests to declare.